Piperazine and piperidine derivatives, their synthesis and use thereof in inhibiting VDAC oligomerization, apoptosis and mitochondria dysfunction

ABSTRACT

Provided herein piperazine and piperidine derivatives, their synthesis and use thereof in inhibiting VDAC oligomerization, apoptosis and mitochondria dysfunction. Also provided methods of treatment of diseases associated with said processes, e.g. Alzheimer&#39;s disease.

This application is the U.S. national phase of International ApplicationNo. PCT/IL2016/051020 filed Sep. 13, 2016, which designated the U.S. andclaims priority to 62/217,986 filed Sep. 14, 2015.

The invention relates to use of small organic compounds interacting withthe Voltage-Dependent Anion Channel (VDAC), reducing its channelconductance and acting as inhibitors of VDAC oligomerization, associatedwith apoptosis induction, for the treatment of diseases associated withenhanced apoptosis. In particular the present invention relates to thecompounds of the general formulae (I) and (II) for the treatment ofenhanced apoptosis-associated diseases, such as neurodegenerative andcardiovascular diseases. The present invention also embracespharmaceutical compositions comprising these compounds and methods ofusing the compounds and their pharmaceutical compositions.

VDAC forms the main interface between mitochondrial and cellularmetabolisms by mediating the fluxes of ions, nucleotides and othermetabolites across the outer mitochondrial membrane (OMM)(Shoshan-Barmatz, V., et. al (2010) VDAC, a multi-functionalmitochondrial protein regulating both cell life and death. MolecularAspects of Medicine 31(3), 227-286; Shoshan-Barmatz, V. and Ben-Hail, D.(2012) VDAC, a multi-functional mitochondrial protein as apharmacological target, Mitochondrion, 12(1):24-34). VDAC has also beenrecognized as a key protein in mitochondria-mediated apoptosis. VDACmediates the release of apoptosis-inducing proteins from mitochondria tothe cytosol and regulates apoptosis via interaction with pro- andanti-apoptotic proteins (Shoshan-Barmatz, V., et. al (2010) VDAC, amulti-functional mitochondrial protein regulating both cell life anddeath. Molecular Aspects of Medicine 31(3), 227-286; Shoshan-Barmatz V.and Golan M. (2012) Mitochondrial VDAC: Function in cell life and deathand a target for cancer therapy, Current Medicinal Chemistry 19(5),714-735).

Piperazine and piperidine are used as essential sub-structure motifs invarious drugs. Piperazine pyrrolidine-2,5-dione derivatives have alsobeen demonstrated as malic enzyme inhibitors (Zhang, Y. John et al.2006. Biorganic & Medicinal Chemistry Letters 16, 525-528).

It has now been found by the present inventors that members of a novelgroup of piperazine- and piperidine-based compounds directly interactwith and have high inhibitory activity of VDAC oligomerization and arethus useful as inhibitors of its channel conductance, it oligomerizationand thereby as inhibitors of the release of apoptogenic proteins fromthe mitochondria, as well as inhibitors of apoptotic cell death or othercell death types as necrosis.

The present invention provides substituted piperazine- andpiperidine-derivatives of general Formula (I)

wherein the groups R¹, R², R³, L¹, L² and A are as defined hereinafter,including the stereoisomers, enantiomers, mixtures thereof and saltsthereof.

Compounds of general Formulae (I) and (II), as defined hereinafter, aresuitable for inhibiting the oligomerization of mitochondrialVoltage-Dependent Anion Channel (VDAC) protein, an early and criticalstep in the progression of apoptosis. Compounds of general Formulae (I)and (II) are also suitable for protecting a cell against apoptosis.Compounds of general Formulae (I) and (II) are further suitable forprotecting a cell against mitochondrial dysfunction associated withapoptosis induction and/or compromised cell energy production, reactiveoxygen radicals (ROS) production and/or alterations in intracellularcalcium concentration.

The invention also relates to processes for preparing a compound ofgeneral Formula (I) according to the invention.

The invention is further directed to pharmaceutical compositionscontaining a compound of Formulae (I) or (II) according to theinvention, as well as to the use of the compounds of Formulae (I) and(II) for preparing a pharmaceutical composition for the treatment ofdiseases and disorders, especially diseases and disorders associatedwith enhanced apoptosis or other cell death types as necrosis.

Other aspects and embodiments of the present invention will becomeapparent to the skilled person from the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates a representative chromatogram and respective massspectra of two peaks of interest relating to Intermediate 1.

FIG. 2a demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to the compound of Formula 2.

FIG. 2b demonstrates a representative NMR spectrum relating to thecompound of Formula 2.

FIG. 3 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 2.

FIG. 4a demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to the compound of Formula 1.

FIG. 4b demonstrates a representative NMR spectrum relating to thecompound of Formula 1.

FIG. 5 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 3.

FIG. 6 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 4.

FIG. 7 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 5.

FIG. 8 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 6.

FIG. 9 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 7.

FIG. 10 demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to Intermediate 8.

FIG. 11a demonstrates a representative chromatogram and respective massspectrum of the peak of interest relating to the compound of Formula 3.

FIG. 11b demonstrates a representative NMR spectrum in deuterated DMSOrelating to the compound of Formula 3.

FIG. 11c demonstrates a representative NMR spectrum in deuterated DMSOand deuterated water (D₂O) relating to the compound of Formula 3.

FIGS. 12a and 12b demonstrate representative NMR spectra in deuteratedDMSO relating to separated single enantiomers of the compound of Formula1 (identified as BGD-4-1 and VBIT-4-2, respectively).

FIG. 13a demonstrates the BRET2 signals in VDAC1 dimerization experimentwith or without the VDAC1 dimerization inhibitor DNDS ((Ben-Hail D,Shoshan-Barmatz V.VAC1-interacting anion transport inhibitors inhibitVDAC1 oligomerization and apoptosis. Biochim Biophys Acta. 2016 July;1863(7 Pt A):1612-2), in absense or presence of selenite.

FIG. 13b demonstrates a representative VDAC1 immunoblot ofelectroblotted gel in VDAC1 oligomerization experiment with or withoutthe VDAC1 dimerization inhibitor DNDS ((Ben-Hail D, Shoshan-BarmatzV.VDAC1-interacting anion transport inhibitors inhibit VDAC1oligomerization and apoptosis. Biochim Biophys Acta. 2016 July; 1863(7Pt A):1612-2), in absense or presence of selenite. VDAC oligomers werestabilized by EGS cross-linking.

FIG. 14a demonstrates a representative VDAC1 immunoblottedelectroblotted gel of VDAC1 oligomerization experiment with or withoutthe single enantiomers or the racemic compound of Formula 1, in presenceof selenite. VDAC1 oligomers were stabilized by EGS cross-linking.

FIG. 14b schematically demonstrates an apoptotic cell death inhibitionas function of concentration of the single enantiomers or the racemiccompound of Formula 1 in presence of selenite.

FIG. 15a demonstrates a representative VDAC1 immunostainedelectroblotted gel in VDAC1 oligomerization experiment with or withoutthe racemic compound of Formula 1, and the compounds of Formulae 2 and10, in presence of selenite.

FIG. 15b demonstrates inhibition of VDAC1 oligomerization as function ofconcentration of the compounds of Formulae 1, 2 and 10, in presence ofselenite. The closed circle (●) indicates compound of Formula 1, theopen circle (∘) indicates compound of Formula 10, and an open square (□)indicates compound of Formula 2.

FIG. 15c schematically demonstrates inhibition of selenite-inducedapoptotic cell death as function of concentration of the compounds ofFormulae 1, 2 and 10. The closed circle (●) indicates compound ofFormula 1, the open circle (∘) indicates compound of Formula 10, and anopen square (□) indicates compound of Formula 2.

FIG. 15d demonstrates a representative VDAC1 immunostainedelectroblotted gel bands of Cyto c and VDAC1 in mitochondria and incytosolic fraction after exposure to selenite and the compounds ofFormulae 1, 2 and 10.

FIG. 15e demonstrates inhibition of selenite-induced Cyto C release as afunction of concentration of the compounds of Formulae 1, 2 and 10. Theclosed circle (●) indicates compound of Formula 1, the open circle (∘)indicates compound of Formula 10, and an open square (□) indicatescompound of Formula 2.

FIG. 16a demonstrates a representative immunostained electroblotted gelof VDAC1 after exposure to the compounds of Formulae 1 and 10 in absenceor presence of cisplatin in SH-SY5Y cells.

FIG. 16b demonstrates a representative immunostained electroblotted gelof VDAC1 after exposure to the compounds of Formulae 1 and 10 inpresence of cisplatin in Bax^(−/−)/Bak^(−/−) MEF cells.

FIG. 16c demonstrates inhibition of apoptotic cell death and of VDAC1dimers formation in the presence of the compounds of Formulae 1 and 10,in presence of cisplatin in SH-SY5Y cells.

FIG. 16d demonstrates inhibition of apoptosis Cyto C release and ofVDAC1 dimers formation in the presence of the compounds of Formulae 1and 10, in presence of cisplatin in Bax^(−/−)/Bak^(−/−) MEF cells.

FIG. 17a demonstrates a representative VDAC1 immunostainedelectroblotted gel of VDAC1 after exposure to increasing concentrationsof the compound of Formula 10 in presence of selenite.

FIG. 17b demonstrates inhibition of apoptosis and VDAC1 dimers formationas function of exposure to increasing concentrations of the compound ofFormula 10 in presence of selenite.

FIG. 17c demonstrates a representative VDAC1 immunostainedelectroblotted gel of VDAC1 after exposure to increasing concentrationsof the compound of Formula 10 in presence of cisplatin.

FIG. 17d demonstrates inhibition of apoptosis and VDAC1 dimers formationas function of exposure to increasing concentrations of the compound ofFormula 10 in presence of cisplatin.

FIG. 17e demonstrates the extent of apoptosis inhibition as function ofVDAC1 dimerization inhibition obtained at the same concentration ofFormula 10 and as induced by either cisplatin (empty square—□) orselenite (solid square—▪).

FIG. 18a demonstrates current through purified VDAC1 channel in presenceof compounds of Formulae 1, 2, and 10.

FIG. 18b demonstrates the fraction of maximal conductance of purifiedVDAC1 in presence of the compounds of the Formulae 1 (solid circle), 2(empty square) or 10 (empty circle), at varying applied voltage.

FIG. 18c demonstrates the bound fraction of the compounds of theFormulae 1, 2 and 10 to the purified VDAC1, as a function of theirconcentration.

FIG. 19a demonstrates the effect of compounds of Formulae 1 and 10 onintracellular calcium ion concentrations in cells treated selenite.

FIG. 19b demonstrates the effect of compounds of Formulae 1 and 10 onmitochondrial membrane potential in cells treated selenite.

FIG. 19c demonstrates the effect of compounds of Formulae 1 and 10 onROS levels in cells treated selenite.

FIG. 19d demonstrates schematically the effect of compounds of Formulae1 and 10 on mitochondrial superoxide levels in cells treated selenite.

FIG. 20A demonstrates the effect of compound of Formula 1 on learningand memory of transgenic mice with Alzheimer's disease like symptomsusing Radial Arm Water Maze test; number of errors is demonstrated asfunction of learning blocks.

FIG. 20B demonstrates the effect of compound of Formula 1 on learningand memory of transgenic mice with Alzheimer's disease like symptomsusing Radial Arm Water Maze test; total time per test is demonstrated asfunction of learning blocks.

According to one aspect of the invention there is provided a compound ofthe general formula (I):

wherein:

A is carbon (C) or nitrogen (N);

R³ is hydrogen or heteroalkyl group; wherein when A is nitrogen (N), R³is absent;

L¹ is a linking group which may be absent or present, but if present isan amino linking group —NR⁴—, wherein R⁴ is hydrogen, a C_(1-n)-alkyl,wherein n is an integer from 2 to 5, inclusive, or a substituted alkylCH₂—R, wherein R is a functional group selected from hydrogen, halo,haloalkyl, cyano, nitro, hydroxyl, alkyl, alkenyl, aryl, alkoxyl,aryloxyl, aralkoxyl, alkylcarbamido, arylcarbamido, amino, alkylamino,arylamino, dialkylamino, diarylamino, arylalkylamino, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonyloxy,arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo,alkylsulfonylamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl,arylsulfinyl or heteroaryl; preferably R⁴ is hydrogen;

R¹ is an aromatic moiety, preferably phenyl, which may be substitutedwith one or more of Z;

Z is independently one or more of functional groups selected from,hydrogen, halo, haloalkyl, haloalkoxy, perhaloalkoxy orC₁₋₂-perfluoroalkoxy, cyano, nitro, hydroxyl, alkyl, alkenyl, aryl,alkoxyl, aryloxyl, aralkoxyl, alkylcarbamido, arylcarbamido, amino,alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino,aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonyloxy,arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo,alkylsulfonylamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl,arylsulfinyl or heteroaryl; preferably Z is C₁₋₂-perfluoroalkoxy;preferably R¹ is a phenyl and Z is trifluoromethoxy; preferably R¹ is aphenyl substituted with one trifluoromethoxy, most preferably at thepara position;

L² is a linking group, such that when A is nitrogen (N), L² is a groupconsisting of 4-10 atoms (apart from hydrogen atoms), optionally forminga closed ring, whereof at least one of the atoms is nitrogen, saidnitrogen forming part of an amide group; preferably said linking groupis selected from the group consisting of an C₄₋₆-alkylamidylene and apyrrolidinylene, said linking group optionally substituted with one ortwo of alkyl, hydroxy, oxo or thioxo group; most preferably L² isselected from butanamidylene, N-methylbutanamidylene,N,N-dimethylbutanamidylene, 4-hydroxybutanamidylene(HO—CH₂—C*H—CH₂—C(O)NH—, wherein the asterisk denotes attachment point),4-oxobutanamidylene, 4-hydroxy-N-methylbutanamidylene,4-oxo-N-methylbutanamidylene, 2-pyrrolidonyle,pyrrolidine-2,5-dionylene, 5-thioxo-2-pyrrolidinonylene and5-methoxy-2-pyrrolidinonylene;

and when A is carbon (C), then L² is C_(1-n) alkylene, wherein n is aninteger from 2 to 4, inclusive; L² is preferably methylene (—CH₂—);

R² is a phenyl or a naphthyl, optionally substituted with halogen,preferably when R² is a phenyl it is substituted with halogen,preferably chlorine, at the para position, preferably when R² isnaphthyl, L² is an alkylene group, preferably —CH₂—;

with a proviso that when A is carbon (C), L¹ is —NR⁴—, R⁴ is hydrogen,and R² is phenyl substituted with chlorine, then L² is notpyrrolidine-2,5-dione.

In one embodiment, when A is nitrogen (N), the linking group L² isselected from the group consisting of an C₄₋₆-alkylamidylene and apyrrolidinylene, said linking group optionally substituted with one ortwo of alkyl, hydroxy, oxo or thioxo group. For example, L² may bebutanamidylene, N-methylbutanamidylene, N,N-dimethylbutanamidylene,4-hydroxybutanamidylene, 4-oxobut-anamidylene,4-hydroxy-N-methylbutanamidylene, 4-oxo-N-methyl-butanamidylene,2-pyrrolidonyle, pyrrolidine-2,5-dionylene, 5-thioxo-2-pyrrolidinonyleneor 5-methoxy-2-pyrrolidinonylene. Preferably, when L² is butanamidylene,N-methylbutanamidylene, N,N-dimethylbutanamidylene,4-hydroxybutanamidylene, 4-oxobut-anamidylene,4-hydroxy-N-methylbutanamidylene or 4-oxo-N-methylbutanamidylene, thenpreferably the carbon in third position (C) of the butanamide moiety isbonded to the nitrogen (N) of the piperazine ring or the piperidine ringand the nitrogen (N) of the butanamide moiety is bonded to R². Forexample, when L² is 2-pyrrolidone, pyrrolidine-2,5-dione,5-thioxo-2-pyrrolidone or 5-methoxy-2-pyrrolidone, then preferably acarbon (C) of the pyrrolidine moiety is bonded to the nitrogen (N) ofthe piperazine ring or the piperidine ring and the nitrogen (N) of thepyrrolidine moiety is bonded to R².

In another embodiment, A is carbon (C), R³ is heteroalkyl and L² ismethylene.

The invention also relates to the stereoisomers, enantiomers, mixturesthereof, and salts, particularly the physiologically acceptable salts,of the compounds of general Formula (I) according to the invention.

According to another aspect of the invention there is provided acompound of the general formula (Ia):

wherein:

A, R³, Z and L¹ are as previously defined in reference to compound ofFormula (I); preferably A is nitrogen (N);

L²′ is a linking group selected from the group consisting of anC₄-alkylamidylene, C₅-alkylamidylene or C₆-alkylamidylene, optionallysubstituted with one or two of alkyl, hydroxy, oxo or thioxo group;preferably L²′ is selected from butanamidylene, N-methylbutanamidylene,N,N-dimethylbutanamidylene, 4-hydroxybut-anamidylene,4-oxobutanamidylene, 4-hydroxy-N-methylbutan-amidylene or4-oxo-N-methylbutanamidylene; most preferably L²′ is4-hydroxybutanamidylene; wherein preferably the carbon (C) at position 3of the alkyl moiety of alkylamidylene L²′ is bonded to the nitrogen (N)of the piperazine ring or of the piperidine ring, and the nitrogen (N)of the butanamide moiety is bonded to the phenyl group; preferably L²′is HO—CH₂—C*H—CH₂—C(O)NH—, wherein the asterisk denotes attachmentpoint;

Y is halogen, preferably chlorine, e.g. at the para position;

or an enantiomer, diastereomer, mixture or salt thereof.

According to another aspect of the invention there is provided acompound of the general formula (Ib):

wherein:

A, R³, and Z are as previously defined in reference to the compound ofFormula (I); preferably A is nitrogen (N);

L¹ is absent;

L²″ is a pyrrolidinylene linking group, optionally substituted with oneor two of alkyl, hydroxy, oxo or thioxo group, preferably L²″ isselected from 2-pyrrolidonylene, pyrrolidine-2,5-dionylene,5-thioxo-2-pyrrolidinonylene and 5-methoxy-2-pyrrolidinonylene; mostpreferably L²″ is pyrrolidine-2,5-dionylene; wherein preferably a carbon(C) at position 4 or the carbon (C) at position 3 of the pyrrolidinylmoiety L²″ is bonded to the nitrogen (N) of the piperazine ring or thepiperidine ring and the nitrogen (N) of the pyrrolidinyl moiety isbonded to the phenyl group substituted with Y;

Y is halogen, preferably chlorine, e.g. at the para position;

provided that when A is carbon (C), L¹ is present and R⁴ is hydrogen,L²″ is not pyrrolidine-2,5-dione.

According to yet another aspect of the invention there is provided acompound of the general formula (Ic):

wherein:

A, R³, and Z are as previously defined in reference to the compounds ofgeneral Formula (I);

L¹ is —NH—;

Y¹ and Y² may be absent or present, but if present are independently ahalogen;

or an enantiomer, diastereomer, mixture or salt thereof.

Preferred compounds of Formula (Ic) are those wherein R³ is—C(O)NHCH₂C(O)OH group, and/or wherein Z is C₁₋₂-alkoxy or halogenatedC₁₋₂-alkoxy, e.g. C₁₋₂-perfluoroalkoxy.

According to another aspect of the invention there is provided acompound of the general formula (Id):

wherein

L² is selected from the group consisting of an C₄₋₆-alkylamidylene (e.g.HO—CH₂—C*H—CH₂—C(O)NH—, wherein the asterisk denotes attachment point),and a pyrrolidinylene (e.g. pyrrolidin-2,5-dionylene), optionallysubstituted with one or two of alkyl, hydroxy, oxo or thioxo group;

Z is haloalkoxy, e.g. C₁₋₂-perfluoroalkoxy, and Y is halogen.

The invention also relates to the stereoisomers, enantiomers, mixturesthereof and salts thereof, of the compounds of general Formulae (Ia),(Ib), (Ic), and (Id), according to the invention.

Table 1 provides non-limiting examples of compound of general Formula(I). It includes compounds as follows:N-(4-chlorophenyl)-4-hydroxy-3-(4-(4-(trifluoromethoxy)phenyl)-piperazin-1-yl)butanamide(Formula 1),1-(4-chlorophenyl)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)pyrrolidine-2,5-dione(Formula 2),1-(naphthalen-1-yl)methyl)-4-(phenylamino)-piperidine-4-carbonyl)glycine(Formula 3),1-(4-chlorophenyl)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)pyrrolidin-2-one(Formula 4),1-(4-chlorophenyl)-5-thioxo-3-(4-(4-(trifluoro-methoxy)phenyl)piperazin-1-yl)pyrrolidin-2-one(Formula 5),1-(4-chlorophenyl)-5-methoxy-4-(4-(4-(trifluoromethoxy)phenyl)-piperazin-1-yl)pyrrolidin-2-one(Formula 6),1-(4-chlorophenyl)-5-thioxo-4-(4-((4-(trifluoromethoxy)phenyl)amino)piperidin-1-yl)pyrrolidin-2-one(Formula 7),4-(4-chlorophenyl)-4-oxo-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanamide(Formula 8),N-(4-chlorophenyl)-4-hydroxy-N-methyl-3-(4-(4-(trifluoro-methoxy)phenyl)piperazin-4-yl)butanamide(Formula 9).

TABLE 1 Description Formula # Structure (according to general Formula(I)) 1

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is phenyl substitutedwith one trifluoromethoxy, L² is 4- hydroxybutanamidylene, the 3^(rd)carbon (C) of the butanamide moiety is bonded to the nitrogen (N) of thepiperazine ring, the nitrogen (N) of the butanamide moiety is bonded toR² and R² is a phenyl substituted with chlorine at the para position[also identified herein as VBIT-4 or as BGD-4] 2

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is phenyl substitutedwith one trifluoromethoxy, L² is pyrrolidine-2,5- dione, the carbon (C)at position 3 of the pyrrolidine moiety is bonded to the nitrogen (N) ofthe piperazine ring, the nitrogen (N) of the pyrrolidine moiety isbonded to R² and R² is a phenyl substituted with chlorine at the paraposition [also identified herein as VBIT-3 or as BGD-3] 3

A is carbon (C), R³ is —C(O)NHCH₂C(O)OH group; L¹ is —NH—, R¹ is aphenyl, L² is methylene and R² is a naphthyl [also identified herein asVBIT-12] 4

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is a phenylsubstituted with one trifluoromethoxy; L² is 2-pyrrolidone, the carbon(C) at position 3 of the pyrrolidone moiety is bonded to the nitrogen(N) of the piperazine ring, the nitrogen (N) of the pyrrolidone moietyis bonded to R² and R² is a phenyl substituted with chlorine at the paraposition [also identified herein as VBIT-5] 5

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is a phenylsubstituted with one trifluoromethoxy, L² is 5-thioxo-2- pyrrolidone,the carbon (C) at position 3 of the pyrrolidine moiety is bonded to thenitrogen (N) of the piperazine ring, the nitrogen (N) of the pyrrolidinemoiety is bonded to R² and R² is a phenyl substituted with chlorine atthe para position [also identified herein as VBIT-6] 6

A is carbon (C), R³ is hydrogen, L¹ is —NH—, R¹ is a phenyl substitutedwith one trifluoromethoxy, L² is 5-methoxy-2- pyrrolidinone, the carbon(C) at position 4 of the pyrrolidine moiety is bonded to the nitrogen(N) of the piperidine ring, the nitrogen (N) of the pyrrolidine moietyis bonded to R² and R² is a phenyl substituted with chlorine at the paraposition [also identified herein as VBIT-9] 7

A is carbon (C), R³ is hydrogen, L¹ is —NH—, R¹ is a phenyl substitutedwith one trifluoromethoxy, L² is 5-thioxo-2- pyrrolidone, the carbon (C)at position 3 of the pyrrolidine moiety is bonded to the nitrogen (N) ofthe piperidine ring, the nitrogen (N) of the pyrrolidine moiety isbonded to R² and R² is a phenyl substituted with chlorine at the paraposition [also identified herein as VBIT-10] 8

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is phenyl substitutedwith one trifluoromethoxy, L² is 4-oxobutanamide, the 3^(rd) carbon (C)of the butanamide moiety is bonded to the nitrogen (N) of the piperazinering, the 4^(th) carbon (C) of the butanamide moiety is bonded to R² andR² is a phenyl substituted with chlorine at the para position [alsoidentified herein as VBIT-7] 9

A is nitrogen (N), R³ is absent, L¹ is absent, R¹ is phenyl substitutedwith one trifluoromethoxy, L² is 4-hydroxy-N- methylbutanamide, the3^(rd) carbon (C) of the butanamide moiety is bonded to the nitrogen (N)of the piperazine ring, the nitrogen (N) of the butanamide moiety isbonded to R² and R² is a phenyl substituted with chlorine at the paraposition [also identified herein as VBIT-8]

Some terms used herein to describe the compounds according to theinvention are defined more specifically below.

The term halogen denotes an atom selected from among F, Cl, Br and I,preferably Cl and Br.

The term heteroalkyl as used herein in reference to R³ moiety of thegeneral Formulae (I), (Ia), (Ib), (Ic), (Id), (II) and (IIa), refers toa saturated or unsaturated group of 3-12 atoms (apart from hydrogenatoms), wherein one or more (preferably 1, 2 or 3) atoms are a nitrogen,oxygen, or sulfur atom, for example an alkyloxy group, as for examplemethoxy or ethoxy, or a methoxymethyl-, nitrile-,methylcarboxyalkylester- or 2,3-dioxyethyl-group; preferably heteroalkylgroup is a chain comprising an alkylene, and at least one of acarboxylic acid moiety, a carbonyl moiety, an amine moiety, a hydroxylmoiety, an ester moiety, an amide moiety. The term heteroalkyl refersfurthermore to a carboxylic acid or a group derived from a carboxylicacid as for example acyl, acyloxy, carboxyalkyl, carboxyalkylester, suchas for example methylcarboxyalkylester, carboxyalkylamide,alkoxycarbonyl or alkoxycarbonyloxy; preferably the term refers to—C(O)NHCH₂C(O)OH group.

The term C_(1-n)-alkyl, wherein n may have a value as defined herein,denotes a saturated, branched or unbranched hydrocarbon group with 1 ton carbon (C) atoms. Examples of such groups include methyl, ethyl,n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl,iso-pentyl, neo-pentyl, tert-pentyl, n-hexyl, iso-hexyl, etc.

The term C_(1-n)-alkoxy, wherein n may have a value as defined herein,denotes an alkyl group as defined herein, bonded via —O— (oxygen)linker.

The term C_(1-n)-perfluoroalkoxy, wherein n may have a value as definedherein, denotes an alkoxy group with hydrogen atoms substituted byfluorine atoms.

The term C_(1-m)-alkylamidyl, wherein m may have a value as definedherein, denotes a group comprising 1 to m carbon (C) atoms and an amidegroup formed by either C_(m-a)alkyl-COOH and H₂N—C_(a)alkyl, orC_(m-a)alkyl-NH₂ and HOOC-C_(a)alkyl, wherein a is smaller than or equalto m. Similarly, the terms C₄-alkylamidylene, C₅-alkylamidylene andC₆-alkylamidylene refer to divalent C_(m)-alkylamidyl groups, wherein mis either 4, 5, or 6, respectively.

The term “VDAC” as used herein, unless the context explicitly dictatesotherwise, refers to Voltage-Dependent Anion Channel protein, to all itsisoforms, e.g. to isoform VDAC1, to isoform VDAC2, or to isoform VDAC3.

According to another aspect of the invention, provided herein is aprocess for the preparation of a compound of Formula (I). The compoundsof Formula (I) according to the invention may be obtained using knownmethods of synthesis. Preferably the compounds are obtained by methodsof preparation that are described more fully hereinafter.

Certain compounds of the general Formula (I), wherein L¹ is absent, andA is nitrogen, may be prepared by coupling an aryl halide of the formulaR¹—X, wherein X is a halogen, preferably bromide, with a mono-protectedpiperazine, e.g. with BOC-protected piperazine, and upon deprotection,reacting with a L²-linker precursor reactive with secondary amines, andsubsequent amidation or transamidation of the L²-linker precursor moietywith a suitable amine of the formula (Y)R²—NH₂. The L²-linker precursormay be an unsaturated C₄₋₆ carboxylic derivative compound, e.g. anunsaturated C₄₋₆ lactone, or a β-, γ-, δ- or ε-unsaturated linear esterof the C₄₋₆ carboxylic acid and a suitable alcohol, e.g. C₁₋₆ alcohol.Alternatively, the deprotected R¹-piperazine may be reacted with asuitable N—R²-pyrrolidenone or N—R²-pyrrolidinene-dione, prepared bygenerally known methodology (e.g. in Synthesis, anticonvulsant activityand 5-HT1A, 5-HT2A receptor affinity of newN-[(4-arylpiperazin-1-yl)-alkyl] derivatives of 2-azaspiro[4.4]nonaneand [4.5]decane-1,3-dione, by Obniska, J.; Kolaczkowski, M.; Bojarski,A. J.; Duszynska, B., European Journal of Medicinal Chemistry (2006),41(7), 874-881).

Compounds of general Formulae (Ia) and (Ib) may be prepared according toa Method (a) shown in Schemes 1 to 3, starting from a compound ofgeneral Formula A, wherein Z is as hereinbefore defined.

Compounds of general Formula C are obtained by reacting a compound ofgeneral Formula A with a piperizine in which one of the nitrogens isprotected with a protecting group, e.g. tert-butyloxycarbonyl protectinggroup (BOC group). The starting compounds of general Formula A is eithercommercially obtainable or may be prepared by using known methods fromcommercially obtainable compounds. The carbon-nitrogen (C—N) couplingreaction is carried out in the presence of a palladium catalyst, such astris(dibenzylideneacetone)dipalladium (Pd₂(dba)₃) that is particularlysuitable. The reaction is carried out in a presence of a bidentatephosphine ligand and a base. Suitable bidentate phosphine ligands arediphenyl-phosphinobinapthyl (BINAP) and diphenylphosphinoferro-cene(DPPF), while BINAP is particularly preferred. Suitable bases includesodium tert-butoxide, potassium tert-butoxide, lithiumbis(trimethylsilyl)-amide, while sodium tert-butoxide is particularlysuitable. The reaction is carried out in a suitable aprotic solvent suchas toluene, tetrahydrofuran (THF), dioxane, but preferably toluene,under nitrogen atmosphere, and at a temperature between 55° C. and 110°C., preferably at a temperature of 65° C. and 110° C., most preferably80° C. and 110° C. Upon completion of the reaction the solvent isevaporated to provide crude compound of Formula C as a residue that maybe used for the next step without further purification.

Compounds of general Formula D are obtained by removal of the protectinggroup, e.g. BOC, in the compound of general Formula C, which can beaccomplished with strong acids such as trifluoroacetic acid, neat or indichloromethane, or with concentrated HCl in methanol or indichloromethane (DCM), while concentrated HCl in DCM is preferred. Thereaction is preferably carried out at room temperature. Upon completionof the reaction the organic phase is discarded and the aqueous phaseevaporated to dryness. The residue is dissolved in a base and a suitablesolvent, such as DCM, dichloroethane or 2-methyltetrahydrofuran (MeTHF),NaOH (2.0 M) and DCM are preferred. Upon completion of the reaction, theorganic solvent phase, e.g. DCM, is collected and concentrated to yieldthe crude product of general Formula D that may be used for the nextstep without further purification.

Compounds of general Formula (Ia) and (Ib) are obtained from compoundsof general Formula D. For example, certain preferred compounds ofgeneral Formula (Ia) may be obtained according to Scheme 2, by reactinga compound of general Formula D with a suitable lactone, e.g.2-furanone, to yield a compound of general Formula E, which is reactedwith a suitable aminophenyl to yield a compound of general Formula (Ia).

For example, certain preferred compounds of general Formula (Ib) may beobtained according to Scheme 3, by reacting a compound of generalFormula D with a suitable pyrrole-dione, e.g1-Phenyl-1H-pyrrole-2,5-dione, which is commercially available or mayreadily be prepared by methods familiar to those skilled in the art, toyield a compound of general Formula (Ib).

Certain compounds of the general Formula (I) wherein R³ is present andis not hydrogen and wherein L¹ is present, can be prepared fromhalogenated compounds of the general formula (Y¹)(Y²)R²-L²X that arecoupled with a protected piperidone in presence of a base, and therecovered ketone is R³-sililated, e.g. nitrilosililated, in presence ofan amine of the general formula R¹-L¹-H (L¹-H being the —NR⁴— group,with R⁴ as defined hereinabove) in acid environment to furnish thecompound of general formula (Y¹)(Y²)R²-L²-N[—CH₂—CH₂—]₂C(R³)L¹-R¹,wherein R³ is the nitrile. Thereafter, the nitrile may be hydrolyzed ina strong acid to a respective amide and further in a strong base to arespective carboxylic acid, which is reacted by peptidic methodology toa protected glycinate ester, finally deprotected to furnish the compoundof formula (I).

Certain preferred compounds of general Formula (Ic) may be preparedaccording to a Method (b) shown in Schemes 4 and 4a, starting from anaphthyl compound of general Formula F, wherein X is halogen, preferablychlorine, p is an integer having a value of 1, 2 or 3 and Y¹ and Y² areas hereinbefore defined.

Generally, compounds of general Formula H are obtained by reacting acompound of general Formula F with a piperidone, preferably4-piperidone, protected with a suitable glycol, e.g. ethylene glycol,followed by deprotection of the ketone. The reaction is carried out in apolar solvent, such as dimethyl formamide (DMF) or tetrahydrofuran(THF), in presence of a base. Suitable base may be a carbonate, e.g.potassium carbonate, sodium carbonate. The reaction may be carried outat a temperature between 0° C. and 60° C., preferably between 15° C. and40° C., most preferably at ambience, e.g. at room temperature. Thereaction may be kept for about 6-18 hours, preferably for about 10-14hours. The product may be purified, e.g. by chromatography, anddeprotected by heating the product under acidic conditions. Thedeprotection may be carried out in a suitable solvent, e.g. an alcohol,such as ethanol, that dissolves the acid used, e.g. hydrochloric acid.

The obtained compound of Formula H may be further reacted with asuitable substituted silane, e.g trimethyl sililonitrile (TMSCN), in apresence of a suitable primary or secondary amine, e.g. aniline,piperidine, ethylamine, propylamine, ethylpropylamine, dipropylamine, ina suitable solvent under acidic conditions, e.g. in acetic acid,trifluoroacetic acid, benzoic acid. The reagents may be combined at atemperature lower than 60° C., preferably lower than 40° C., furtherpreferably between 0° C. and 40° C. and most preferably between 10° C.and 20° C. The reaction may be kept for about 6-18 hours, preferably forabout 10-14 hours. After neutralizing the acid, the reaction mixture maybe extracted into an apolar solvent, such as dichloromethane, to yieldthe nitrile compound of formula J, which may be used without furtherpurification.

The nitrile compounds of formula J may be further converted into thecompounds of general Formula (Ic) according to the Scheme 4a below:

The compound of the formula K is prepared by hydrolyzing the nitrilecompound of formula J in a strong acid, e.g. in concentrated sulfuricacid, nitric acid, hydrobromic acid (HBr) and hydrochloric acid (HCl).The reaction may be kept for about 6-18 hours, preferably for about10-14 hours. After neutralization of the reaction mixture, the compoundof the formula K can be purified, e.g. by reverse-phase preparativeHPLC.

The compound of the formula K may be further hydrolyzed into thecompound of formula N, e.g. with potassium hydroxide in a polar solvent,e.g. in ethylene glycol. The reagents may be combined at a temperaturebetween 110° C. and 170° C. and most preferably between 140° C. and 160°C. The reaction may be kept for about 6-18 hours, preferably for about10-14 hours. After cooling of the reaction mixture, the compound of theformula N can be purified, e.g. by reverse-phase preparative HPLC. Thecompound of formula N may then be reacted with methyl glycinate in DMFin presence of a coupling agent, e.g.1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-pyridinium-3-oxidhexafluorophosphate (known as HATU) and diisopropyl ethylamine, forabout 6-18 hours, preferably for about 10-14 hours, and purified, e.g.by reverse-phase preparative HPLC, to furnish the compound of formula Q.Additional coupling agents that may be used areN,N′-dicyclohexylcarbodiimide (DCC),3-(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine (EDC),3-[bis(dimethylamino)-methyliumyl]-3H-benzotriazol-1-oxidehexafluorophosphate (HBTU),1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyrid-inium3-oxid hexafluorophosphate (HATU),1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]-methanaminiumhexafluorophosphate (COMU).

Finally, the compound of formula Q may be hydrolyzed with a base, e.g.lithium hydroxide, to release the methyl ester and furnish the crudecompound of the general Formula (Ic), e.g. in an aprotic solvent, suchas tetrahydrofuran, for about 6-18 hours, preferably for about 10-14hours. The reaction mixture may then be neutralized to pH about 7, andthen purified, e.g. by a preparative HPLC, to furnish the compound ofthe general Formula (Ic).

Compounds of general formula (Id) are prepared according to the methoddescribed for the compounds of general formulae (Ia) and (Ib).

In the reactions described hereinabove, any reactive group such as forexample an amino, alkylamino, hydroxy or carboxy group, may be protectedduring the reaction by conventional protecting groups which are cleavedafter the reaction, by methods known in the art.

The invention also relates to the stereoisomers, such as diastereomersand enantiomers, mixtures and salts, particularly the physiologicallyacceptable salts, of the compounds of general Formulae (I), (Ia), (Ib),(Ic), and (Id), and of the compounds of structural formulae 1, 2, 3, 4,5, 6, 7, 8 and 9.

The compounds of general Formulae (I), (Ia), (Ib), (Ic), and (Id), orintermediate products in the synthesis of compounds of general Formulae(I), (Ia), (Ib), (Ic), and (Id), may be resolved into their enantiomersand/or diastereomers on the basis of their physical-chemical differencesusing methods known in the art. For example, cis/trans mixtures may beresolved into their cis and trans isomers by chromatography. Forexample, enantiomers may be separated by chromatography on chiral phasesor by recrystallisation from an optically active solvent or byenantiomer-enriched seeding.

The compounds of general Formulae (I), (Ia), (Ib), (Ic), and (Id), andthe compounds of structural formulae 1, 2, 3, 4, 5, 6, 7, 8 and 9, maybe converted into the salts thereof, particularly physiologicallyacceptable salts for pharmaceutical use. Suitable salts of the compoundsof general Formulae (I), (Ia), (Ib), (Ic), and (Id), and of thecompounds of structural formulae 1, 2, 3, 4, 5, 6, 7, 8 and 9, may beformed with organic or inorganic acids, such as, without being limitedto hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid,lactic acid, acetic acid, succinic acid, citric acid, palmitic acid ormaleic acid. Compounds of general Formulae (I), (Ia), (Ib), (Ic) and(Id), containing a carboxy group, may be converted into the saltsthereof, particularly into physiologically acceptable salts forpharmaceutical use, with organic or inorganic bases. Suitable bases forthis purpose include, for example, sodium hydroxide, potassiumhydroxide, ammonium hydroxide, arginine or ethanolamine.

According to another aspect provided herein are uses of the compounds ofgeneral Formulae (I), (Ia), (Ib), (Ic), (Id), (II) and (IIa), such as,without being limited to, the compounds of structural formulae 1, 2, 3,4, 5, 6, 7, 8, 9, 10 and 11, for example as oligomerization inhibitorsof Voltage-Dependent Anion Channel (VDAC).

Another aspect of the invention relates to the use of compound accordingto general Formula (II) as defined below, in the preparation ofmedicaments for treatment of diseases as described herein.

wherein:

A is carbon (C) or nitrogen (N);

R³ is hydrogen or heteroalkyl group comprising 3-12 atoms other thanhydrogen, wherein at least one is a heteroatom, preferably selected fromnitrogen, sulfur and oxygen; wherein when A is nitrogen (N), R³ isabsent;

L¹ is a linking group which may be absent or present, but if present isselected from an amino linking group —NR⁴—, wherein R⁴ is hydrogen; aC_(1-n)-alkylene, wherein n is an integer from 2 to 5, inclusive; or asubstituted alkyl, CH₂—R wherein R is a functional group selected fromhydrogen, halo, haloalkyl, cyano, nitro, hydroxyl, alkyl, alkenyl, aryl,alkoxyl, aryloxyl, aralkoxyl, alkylcarbamido, arylcarbamido, amino,alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino,aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonyloxy,arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo,alkylsulfonylamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl,arylsulfinyl or heteroaryl; preferably R⁴ is hydrogen, optionally L¹forming a ring with R³;

R¹ is an aromatic moiety, preferably phenyl, which may be substitutedwith one or more of C₁₋₂-alkoxy, C₁₋₂-perfluoroalkoxy; preferably R¹ isa phenyl substituted trifluoromethoxy; preferably R¹ is a phenylsubstituted with one trifluoromethoxy, preferably at the para position;

L² is a linking group consisting of 4-10 atoms, preferably of 5-6 atoms,optionally forming a closed ring, whereof at least one of the atoms isnitrogen, said nitrogen forming part of an amide group, preferably saidlinking group is selected from the group consisting of anC₄₋₆-alkylamidylene or a pyrrolidinylene, said linking group optionallysubstituted with one or two of alkyl, hydroxy, oxo or thioxo group; morepreferably L² is selected from butanamidylene, N-methylbutanamidylene,N,N-dimethylbutanamidylene, 4-hydroxybutanamidylene,4-oxobutanamidylene, 4-hydroxy-N-methylbutanamidylene,4-oxo-N-methylbutanamidylene, 2-pyrrolidonylene,pyrrolidine-2,5-dionylene, 5-thioxo-2-pyrrolidinonylene and5-methoxy-2-pyrrolidinonylene; or L² is C_(1-n), alkylene, wherein n isan integer between 2 and 5, inclusive, preferably methylene (—CH₂—);said linking group L² bonds piperidine or piperazine moiety at nitrogen(N) atom; and

R² is an aryl, preferably a phenyl or a naphthyl, optionally substitutedwith halogen, optionally when R² is a phenyl it is substituted withhalogen, preferably chlorine, preferably at the para position, furtheroptionally when R² is naphthyl, L² is an alkylenyl group, preferablymethylene (—CH₂—).

Another aspect of the invention relates to the use of compound accordingto general Formula (IIa) as defined below, in the preparation ofmedicaments for treatment of diseases as described herein.

wherein:

A is carbon (C);

R³ is hydrogen or heteroalkyl chain comprising 3-12 atoms, wherein atleast one is a heteroatom, selected from nitrogen, sulfur and oxygen;

L¹ is a linking group which is an amino linking group —NR⁴—, wherein R⁴is hydrogen, C_(1-n)-alkyl, wherein n is an integer from 2 to 5,inclusive, and CH₂—R, wherein R is a functional group selected fromhydrogen, halo, haloalkyl, cyano, nitro, hydroxyl, alkyl, alkenyl, aryl,alkoxyl, aryloxyl, aralkoxyl, alkylcarbamido, arylcarbamido, amino,alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino,aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonyloxy,arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo,alkylsulfonylamido, alkylsulfonyl, arylsulfonyl, alkylsulfinyl,arylsulfinyl or heteroaryl;

when R³ is hydrogen, then L¹ is —NR⁴—; when R³ is heteroalkyl groupcomprising 3-12 atoms, then L¹ is forming a ring with R³;

R¹ is an aromatic moiety, which is optionally substituted with one ormore of C₁₋₂-alkoxy, e.g. haloalkoxy, such as C₁₋₂-perfluoroalkoxy;

L² is a linking group consisting of 4-10 atoms, optionally forming aclosed ring, whereof at least one of the atoms is nitrogen, saidnitrogen forming part of an amide group or L² is C₁₋₂ alkyl or C_(1-n)alkylene, wherein n is an integer between 2 and 5, inclusive; saidlinking group L² bonds piperidine or piperazine moiety at nitrogen (N)atom; preferably, L² is selected from butanamidylene,N-methylbutanamidylene, N,N-dimethylbutanamidylene,4-hydroxybutanamidylene, 4-oxobutanamidylene,4-hydroxy-N-methylbutanamidylene, 4-oxo-N-methylbutanamidylene,2-pyrrolidonylene, pyrrolidine-2,5-dionylene,5-thioxo-2-pyrrolidinonylene and 5-methoxy-2-pyrrolidinonylene; and

R² is an aryl, optionally substituted with halogen, optionally when R²is a phenyl it is substituted with halogen, further optionally when R²is naphthyl, L² is an alkylenyl group. In a specific embodiment, R³ ishydrogen, L¹ is —NH—, and R¹ is a phenyl substituted withtrifluoromethoxy.

The invention also relates to use of the stereoisomers, enantiomers,mixtures thereof, and salts, particularly the physiologically acceptablesalts, of the compounds of general Formulae (II) and (IIa).

In a specific embodiment, there is provided use of compounds accordingto the general Formulae (II) and (IIa), having the structural Formulae10 and 11:

The compound of Formula 10 is also identified herein as AKOS022 orAKOS022075291.

The compound of Formula 11 is also identified herein as DIV 00781.

The compounds of general Formulae (II) and (IIa), such as, without beinglimited to, the compounds of structural formulae 1, 2, 3, 4, 5, 6, 7, 8,9, 10 and 11, may be converted into the salts thereof, particularlyphysiologically acceptable salts for pharmaceutical use. Suitable saltsof the compounds of general Formulae (II) and (IIa), such as, withoutbeing limited to, the compounds of structural formulae 1, 2, 3, 4, 5, 6,7, 8, 9, 10 and 11, may be formed with organic or inorganic acids, suchas, without being limited to hydrochloric acid, hydrobromic acid,sulphuric acid, phosphoric acid, lactic acid, acetic acid, succinicacid, citric acid, palmitic acid or maleic acid. Compounds of generalFormulae (II) and (IIa) containing a carboxy group, may be convertedinto the salts thereof, particularly into physiologically acceptablesalts for pharmaceutical use, with organic or inorganic bases. Suitablebases for this purpose include, for example, sodium salts, potassiumsalts, arginine salts, ammonium salts, or ethanolamine salts.

The compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), (II) and(IIa), especially the specific compounds of Formulae 1, 2, 3, 10 and 11,are inhibitors of Voltage-Dependent Anion Channel (VDAC) oligomerizationand apoptosis. The effect of the compounds of the invention and of thespecific compounds of formulae 1, 2, 3, 10 and 11 on VDAColigomerization, i.e. their ability to inhibit VDAC oligomerization, isdetermined by Bioluminescence Resonance Energy Transfer (BRET2)technology that allows to directly monitor the oligomeric state of VDACmolecules in the native membrane in cells in live. BRET2 screening maybe carried out as described in the art (Keinan et al., (2010)Oligomerization of the mitochondrial protein voltage-dependent anionchannel is coupled to the induction of apoptosis. Mol Cell Biol 30,5698-5709).

The compounds of general Formula (II) and (IIa) have ICso values forseveral activities in the range from 0.1 μM to 10 μM.

The direct interaction between VDAC and the compounds of generalFormulae (I), (Ia), (Ib), (Ic), (Id), (II) and (IIa), especially thespecific compounds of Formulae 1, 2, 3, 10 and 11, may be measured byassessing VDAC channel conductance, following its reconstitution into aplanar lipid bilayer (PLB). The compounds of the general Formulae (I),(Ia), (Ib), (Ic), (Id), (II) and (IIa), interact with VDAC and reduceits channel conductance. To obtain quantitative parameters of thisinteraction and derive a dissociation constant, e.g. a microscalethermophoresis (MST) interaction assay may be performed. In this manner,dissociation values are derived from the curves showing the affinity ofthe compounds of the invention to VDAC.

The compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), (II) and(IIa), especially the specific compounds of Formulae 1, 2, 3, 10 and 11,were found to protect cells against apoptotic cell death. The ability ofthe compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), (II) and(IIa) to inhibit apoptosis may be analyzed by annexin-V/propidium iodide(PI) staining and flow cytometry.

One of the ways cell apoptosis is activated is by release of Cytochromec (Cyto c) from the mitochondria into cytosol. The compounds of generalFormulae (I), (Ia), (Ib), (Ic), (Id), (II) and (IIa), especially thespecific compounds of Formulae 1, 2, 3, 10 and 11, were found to inhibitthe release of Cyto c from mitochondria, as induced by apoptosisstimuli, thereby protecting cells against apoptotic cell death. Cyto creleased to the cytosol may be analyzed by immunoblotting using Cytoc-specific antibodies.

Apoptosis induction was shown to disrupt cell Ca²⁺ homeostasis andenergy production. Indeed, many anti-cancer drugs and other cytotoxicagents, such as thapsigargin, staurosporine, As₂O₃, and selenite, induceapoptotic cell death, as well as disrupt cell Ca²⁺ homeostasis (Keinanet al., (2013) The role of calcium in VDAC1 oligomerization andmitochondria-mediated apoptosis. Biochim Biophys Acta 1833, 1745-1754)).The compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), (II) and(IIa), especially the specific compounds of Formulae 1, 2, 3, 10 and 11,were found to inhibit the elevation in intracellular calcium ionsconcentration ([Ca²⁺]i) elicited by apoptosis stimuli. Increased [Ca²⁺]iis associated with an increase in mitochondrial Ca²⁺, a process expectedto lead to dissipation of the mitochondrial potential (mΔΨ) (Baumgartneret al., (2009) Calcium elevation in mitochondria is the main Ca²⁺requirement for mitochondrial permeability transition pore (mPTP)opening. J Biol Chem 284, 20796-20803). The compounds of generalFormulae (I), (Ia), (Ib), (Ic), (Id), (II) and (IIa), especially thespecific compounds of Formulae 1, 2, 3, 10 and 11, were found to beeffective in preventing the decrease in mΔΨ elicited by a cytotoxicagent. The ability of the compounds of the invention to prevent thedecrease in mΔΨ induced by cytotoxic agent may be measured usingtetramethylrhodamine methylester (TMRM).

The compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), (II) and(IIa), especially the specific compounds of Formulae 1, 2, 3, 10 and 11,are inhibitors of overall cellular reactive oxidative species (ROS)production and of mitochondrial ROS production. The effect of thecompounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), (II) and(IIa) especially the specific compounds of Formulae 1, 2, 3, 10 and 11,on inhibition of cellular ROS production may be determined by 2′,7′dichlorodihydrofluorescein (DCF) fluorescence. The effect of thecompounds of general Formulae (I), (Ia), (Ib), (Ic), (Id) and (II),especially the specific compounds of Formulae 1, 2, 3, 10 and 11, asinhibitors of mitochondrial ROS production may be measured by MitoSOXRed, a mitochondrial superoxide indicator.

In view of the ability of the compounds of general Formulae (I), (Ia),(Ib), (Ic), (Id), (II) and (IIa), especially the specific compounds ofFormulae 1, 2, 3, 10 and 11, to inhibit one or more of theoligomerization of VDAC and/or of the channel ion conductance of VDAC orapoptosis and/or the release of Cyto c from mitochondria and/or theelevation in intracellular calcium concentration ([Ca²⁺]i) elicited by acytotoxic agent and/or production of reactive oxidative species (ROS)the compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), (II) and(IIa), especially the specific compounds of Formulae 1, 2, 3, 10 and 11,and the pharmaceutically acceptable salts thereof, may be suitable fortreating and/or preventing all those conditions or diseases that can beinfluenced by inhibiting one or more of oligomerization of VDAC,apoptosis, release of Cyto c from mitochondria, elevation inintracellular calcium concentration ([Ca²⁺]i) and production of reactiveoxidative species (ROS). Therefore the compounds according to generalFormulae (I), (Ia), (Ib), (Ic), (Id), (II) and (IIa), especially thespecific compounds of Formulae 1, 2, 3, 10 and 11, and thepharmaceutically acceptable salts thereof, are particularly suitable forthe prevention or treatment of diseases or conditions associated withenhanced apoptosis, such as, without being limited to, neurodegenerativeand cardiovascular diseases and disorders. The compounds of generalFormulae (I), (Ia), (Ib), (Ic), (Id), (II) and (IIa), especially thespecific compounds of Formulae 1, 2, 3, 10 and 11, and thepharmaceutically acceptable salts thereof, are also particularlysuitable for the prevention or treatment of diseases or conditions suchas Alzheimer's disease, Parkinson's disease, cardiac hypertrophy, heartfailure, myocardial infarction, ischemia/reperfusion injury, apoptosis,and autophagy of cardiac myocytes, atrial fibrillation (AF), cardiacarrhythmia, and related diseases. It has been found that the treatmentwith a compound of Formula 1 improved the learning and memory task ofAlzheimer's disease-like transgenic mice, to resemble those of wild typemice, as described in the Examples below.

The compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id), (II) and(IIa), such as, without being limited to, the compounds of structuralformulae 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11, especially the specificcompounds of Formulae 1, 2, 3, and 11, and the pharmaceuticallyacceptable salts thereof, may be formulated in a pharmaceuticalcomposition, optionally comprising other active substances, and one ormore of inert conventional excipients, as known to the skilled artisans.The pharmaceutical compositions may be prepared according to the generalguidance provided in the art, e.g. by Remington, The Science andPractice of Pharmacy (formerly known as Remington's PharmaceuticalSciences), ISBN 978-0-85711-062-6. The pharmaceutical compositions, e.g.in the form solid dosage forms, topical dosage form, and/or parenteraldosage forms, e.g. tablets, capsules, creams, ointments, patches,injections, and others as known in the art constitute another aspect ofthe invention.

Particularly, the compounds of general Formulae (I), (Ia), (Ib), (Ic),(Id), (II) and (IIa), particularly of structural formulae 1, 2, 3, 4, 5,6, 7, 8, 9, 10 and 11, especially the specific compounds of Formulae 1,2, 3, 10 and 11, and the pharmaceutically acceptable salts thereof, maybe formulated as nanoparticles. The nanoparticles may be prepared inwell-known polymers, e.g. polylactic-co-glycolic acid, e.g, as describedin H. K. Makadia, S. J. Siegel, Poly Lactic-co-Glycolic Acid (PLGA) asBiodegradable Controlled Drug Delivery Carrier, Polymers (Basel), 3(2011) 1377-1397; and others. Generally, the compounds may beco-dissolved with the polymer in a suitable organic solvent, and theorganic phase may be then dispersed in an aqueous phase comprisingstabilizers and/or surface active agents. The stabilizers may be, e.g.polyvinyl alcohol, with molecular weights from about 89000 to 98000, andhydrolysis degree from about 99%. Upon evaporation of organic solventfrom the aqueous phase, the nanoparticles may be purified, e.g. bycentrifugation and washing.

The encapsulated compounds, e.g. in form of nanoparticles, of generalFormulae (I), (Ia), (Ib), (Ic), (Id), (II) and (IIa), such as, withoutbeing limited to, the compounds of structural formulae 1, 2, 3, 4, 5, 6,7, 8, 9, 10 and 11, especially the specific compounds of Formulae 1, 2,3, 10 and 11, and the pharmaceutically acceptable salts thereof, may beadvantageously used in various routes of administration. Intranasalroute may be suitable mode of administration in this regard.Alternatively, the nanoparticles may be administered systemically toaccumulate in cancerous tissues.

The dose of compounds of general Formulae (I), (Ia), (Ib), (Ic), (Id),(II) and (IIa), such as, without being limited to, the compounds ofstructural formulae 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11, especially thespecific compounds of Formulae 1, 2, 3, and 11, and the pharmaceuticallyacceptable salts thereof, required to achieve treatment or prevention ofa disease or a disorder or a condition usually depends on thepharmacokinetic and pharmacodynamic properties of the compound which isto be administered, the patient, the nature of the disease, disorder orcondition and the method and frequency of administration. Suitabledosage ranges for compounds of general Formulae (I), (Ia), (Ib), (Ic),(Id), (II) and (IIa), such as, without being limited to, the compoundsof structural formulae 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11, especiallythe specific compounds of Formulae 1, 2, 3, 10 and 11, and thepharmaceutically acceptable salts thereof, may be from 1.0 to 100 mg/kgbody weight.

Compounds of Formulae (I), (Ia-d), (II) and (IIa), bearing at least onefluorine atom, e.g. as part of trifluoromethyl group, such as, withoutbeing limited to, the compounds of structural formulae 1, 2, 4, 5, 6, 7,8, 9, and 10, especially the specific compounds of Formulae 1, 2, and10, may be suitable as diagnostic agents for Positron EmissionTomography. For this purpose these compounds may be modified orsynthesized with ¹⁸F isotope, as known in the art. These 18-fluorinatedcompounds may be suitable for imaging of VDAC overexpression in livingorganisms. Particularly, 18-fluorinated compounds of the generalFormulae (I), (Ia), (Ib), (Ic), (Id), (II) and (IIa) may be used todetect pathological conditions or changes due to treatments,particularly for early detection of conditions, e.g. Alzheimer'sDisease, cardiovascular diseases, changes of VDAC expression pattern inpancreatic beta cells, and other tissues.

Accordingly, in another aspect there is provided a method for preventingor treating a disease selected from the list consisting ofneurodegenerative diseases and disorders, cardiovascular diseases anddisorders, Alzheimer's disease, Parkinson's disease, cardiachypertrophy, heart failure, myocardial infarction, ischemia/reperfusioninjury, apoptosis, autophagy of cardiac myocytes, atrial fibrillation(AF), and cardiac arrhythmia, comprising administering to the subject inneed thereof a therapeutically effective amount of a compound of generalformulae (Ia), (Ib), (Ic), (Id), (II) and (IIa), as defined herein.

EXAMPLES

Materials

Carbonyl cyanide m-chlorophenyl hydrazone (CCCP),carboxymethyl-cellulose (CMC), cisplatin, cytochalasin B, dimethylsulfoxide (DMSO), DL-dithiothreitol (DTT), EDTA, HEPES, leupeptine,phenylmethylsulfonyl fluoride (PMSF), N-decane, sodium selenite, soybeanasolectin, staurosporine (STS), tetramethylrhodamine methylester (TMRM)and Tris were purchased from Sigma (St. Louis, Mo.).N,N-Lauryl-(dimethyl)-amineoxide (LDAO) was obtained from Fluka (Bucks,Switzerland). Coelenterazine (Deep Blue C [DBC]) was obtained fromBioline (Taunton, Mass.). Hydroxyapatite (Bio-Gel HTP) was procured fromBio-Rad Laboratories (Hercules, Calif.). Digitonin came fromCalbiochem-Novobiochem (Nottingham, UK). Celite was purchased from theBritish Drug Houses (London, UK). Rabbit monoclonal antibodies againstVDAC1 (ab154856) and mouse monoclonal antibodies against GAPDH (ab9484)came from Abcam (Cambridge, UK). Monoclonal antibodies against actinwere obtained from Millipore (Billerica, Mass.) and anti-Cytochrome cantibodies (556433) were obtained from BD Bioscience (San Jose, Calif.).Polyclonal anti-AIF (Apoptosis Inducing Factor) antibodies came from R&DSystems (Minneapolis, Minn.). Fluo-4-AM™ (CAS name/number: Glycine,N-[4-[6-[(acetyloxy)methoxy]-2,7-difluoro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-,(acetyloxy)methylester 273221-67-3), carboxy-H2DCFDA™(5-(and-6)-carboxy-2″,7″-dichlorodihydrofluorescein diacetate(carboxy-H2DCFDA) and MitoSOX™ Red (red mitochondrial superoxideindicator) were acquired from Invitrogen (Grand Island, N.Y.).Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbitantibodies were obtained from Promega (Madison, Wis.). Ethylene glycolbis[succinimidylsuccinate] (EGS) was obtained from Pierce (Rockford,Ill.). Annexin V-fluorescein isothiocyanate (FITC) was from Enzo LifeSciences (Lausen, Switzerland). Dulbecco's modified Eagle's medium(DMEM) and the supplements fetal bovine serum (FBS), L-glutamine andpenicillin-streptomycin were purchased from Biological Industries(Beit-Haemek, Israel). The compound of Formula 10 (AKOS022) was obtainedfrom AKosConsulting & Solutions GmbH (Germany), under catalogue numberAKOS022075291. Polylactic-co-glycolic acid(poly-D,L-lactide-co-glycolide, 30,000-60,000 Da) was obtained fromSigma. Polyvinyl alcohol, Mw 89000-98000, hydrolysis degree 99%, wasprovided by Sigma.

Methods

LC-MS Analysis

The chromatography was performed using a regular Bridge C18 column4.6×50 mm, 3.5 μm, kept at 40° C. The materials were eluted at 2 mL/min,with mixture of 0.01 M aqueous solution of ammonium carbonate andacetonitrile, with acetonitrile ramping from 5% to 100% for periods asdescribed below and eluting with 100% acetonitrile, with detection atthe target mass.

Tissue Culture

HEK-293, HeLa, SH-SY5Y and K-Ras-transformed Bax^(−/−)/Bak^(−/−)mouseembryonic fibroblast (MEF) cell lines were grown at 37° C. under anatmosphere of 95% air and 5% CO₂ in DMEM supplemented with 10% FBS, 2 mML-glutamine, 1000 U/ml penicillin and 1 mg/ml streptomycin. T-REx-293cells (HEK cells stably containing the pcDNA6/TR regulatory vector andthus expressing the tetracycline repressor; Invitrogen) stablyexpressing hVDAC1-shRNA and showing low (10-20%) endogenous VDAC1expression (referred to herein as T-REx-pS10) were grown under the sameconditions as HEK-293 cells, with an addition of 5 μg/ml blasticidin.

VDAC-1 Cross-Linking

Appropriate test cells (2.5-3 mg/ml) in PBS, were harvested after thetreatment and incubated with the cross-linking reagent EGS at a ratio of100-300 μM for sample of 2-3 mg protein/ml concentration (pH 8.3) for 15minutes. Protein concentration was determined according to Lowry assay.Samples (60-80 μg protein) were subjected to SDS-PAGE by a standardtechnique and immunoblotting was performed using anti-VDAC1 antibodies.Gels were electro-transferred onto nitrocellulose membranes forimmuno-staining. The membranes were incubated with a blocking solutioncontaining 5% non-fat dry milk and 0.1% Tween-20 in Tris-bufferedsaline, followed by incubation with monoclonal anti-VDAC1,anti-Cytochrome c, anti-AIF or anti-actin antibodies. Membranes werethen incubated with HRP-conjugated anti-mouse or anti-rabbit IgG(1:10,000), serving as secondary antibodies. Antibody labeling wasdetected by chemiluminiscence. To detect VDAC1 oligomers, membranes weretreated with 0.1 M glycine, pH 2.0, prior to immunoblotting and washedseveral times with 0.1% Tween-20 in Tris-buffered saline. Quantitativeanalysis of immuno-reactive VDAC1 dimer, trimer and multimer bands wasperformed using FUSION-FX (Vilber Lourmat, France).

VDAC1 Purification

Briefly, rat liver mitochondria (5 mg/ml) in 10 mM Tris-HCl, pH 7.2,were incubated with 2% LDAO at 0° C. for 20 min, followed bycentrifugation (30 min, 14,000 g) and the obtained supernatant wasloaded onto a dry celite:hydroxyapatite (2:1) column. VDAC1 was elutedwith a solution containing 2% LDAO, 10 mM Tris-HCl, pH 7.2, 50 mM NaCl,and 20 to 22 mM NaH₂PO₄, with VDAC1 detection by Coomassie bluestaining. The VDAC1-containing fractions were dialyzed against 10 mMTris-HCl, pH 7.2, and subjected to a second chromatography step on acarboxymethyl-cellulose (CMC) column from which VDAC1 was eluted with asolution containing 10 mM Tris-HCl, pH 7.2, 0.1% LDAO and 500 mM NaCl,detected as above. The VDAC1-containing fractions were collected andused for VDAC1 channel conductance and MST assays.

VDAC1 Channel Conductance

The reconstitution of purified rat VDAC1 into a planar lipid bilayer(PLB) and subsequent single and multiple channel current recordings anddata analysis were carried out as follows. Briefly, the PLB was preparedfrom soybean asolectin dissolved in n-decane (30 mg/ml). Purified VDAC1(1 ng) was added to the chamber defined as the cis side containing 1 MNaCl, 10 mM Hepes, pH 7.4. Currents were recorded under voltage-clampusing a Bilayer Clamp BC-535B amplifier (Warner Instrument, Hamden,Conn.). The currents, measured with respect to the trans side of themembrane (ground), were low-pass-filtered at 1 kHz and digitized onlineusing a Digidata1440-interface board and pClampex 10.2 software (AxonInstruments, Union City, Calif.).

Microscale Thermophoresis (MST) Analysis

MST analysis was performed using a NanoTemper Monolith NT.115 apparatus.Briefly, purified VDAC1 10 μM was fluorescently labeled usingNanoTempers Protein labeling kit BLUE according to manufacturesinstructions (L001, NanoTemper Technologies). A constant concentrationof the protein was incubated with different concentrations of the testedinhibitor in PBS. Afterwards, 3-5 μl of the samples were loaded into aglass capillary (Monolith NT Capillaries) and thermophoresis analysiswas performed (LED 20%, IR laser 20%).

Measurement of Superoxide Generation

ROS (reactive oxygen species) production was monitored using the oxidantsensitive dye DCFDA (2′,7′-dichlorofluorescein diacetate) fluorescentprobe, a cell-permeable indicator of ROS, which is converted by H₂O₂ andperoxidases to the DCF (2′,7′-dichlorofluorescein) fluorescent derivate.Briefly, untreated and treated cells were incubated with DCFDA (4 μM)for 30 minutes. For mitochondrial accumulated ROS, MitoSOX Red (4 μM),mitochondrial superoxide indicator for live-cell imaging was usedaccording to the manufacturer's protocol (Invitrogen, Grand Island,N.Y.). Fluorescence was measured using a FACSCalibur™ flow cytometersoftware (BD Biosciences, Franklin Lakes, N.J.).

Mitochondrial Membrane Potential Determination

Mitochondrial membrane potential (mΔΨ) was determined using TMRM(Tetramethylrhodamine, methyl ester, perchlorate), a potential-sensitivedye, and a plate reader. HEK-293 cells were treated with the testedcompounds and an apoptotic inducer and subsequently incubated with TMRM(0.5 μM, 20 min). The cells were then washed twice with PBS and examinedwith FACSCaliburμ flow cytometer software (BD Biosciences, FranklinLakes, N.J.). CCCP (carbonyl cyanide m-chlorophenyl hydrazine)-mediatedΔΨ dissipation served as control.

Cellular Ca²⁺ Concentration Analysis

Fluo-4 AM™ was used to monitor changes in cytosolic Ca²⁺ levels. Cells,e.g. HeLa cells (1×10⁶ cells/ml) were harvested after the treatment,collected (1,500×g relative centrifugal force (RCF)) (for 10 min) washedwith HBSS (Hanks' Balanced Salt Solution) buffer, pH 7.3-7.4 (5.33 mMKCl, 0.44 mM KH₂PO₄, 138 mM NaCl, 4 mM NaHCO₃, 0.3 mM Na₂HPO₄, 5.6 mMglucose, 0.03 mM phenol red) supplemented with 1.8 mM CaCl₂ (HBSS⁺) andincubated with 2.5 μM Fluo-4 AM™ in 200 μl (HBSS⁺) buffer in the darkfor 30 min at 37° C. After washing the remaining dye, the cells wereincubated with 200 μl (HBSS⁺) buffer and changes in cellular free Ca²⁺concentration were measured immediately with FACS analysis. At least10,000 events were recorded on the FL1 detector, represented as ahistogram, and analyzed with FACS Calibur flow cytometry software.Positive cells showed a shift to an enhanced level of green fluorescence(FL1).

Cytochrome c Release from Mitochondria

Cells treated with apoptosis inducers in the absence or presence of thetested compounds were harvested, washed twice with PBS, pH 7.4 andgently resuspended at 6 mg/ml in ice-cold buffer (100 mM KCl, 2.5 mMMgCl₂, 250 mM sucrose, 20 mM HEPES/KOH pH 7.5, 0.2 mM EDTA, 1 mMdithiothreitol, 1 μg/ml leupeptin, 5 mg/ml cytochalasin B and 0.1 mMPMSF) containing 0.025% digitonin and incubated for 10 min on ice.Samples were centrifuged at 10,000×g (relative centrifugal force—RCF) at4° C. for 5 min to obtain supernatants (cytosolic extracts free ofmitochondria) and pellet (fraction that contains mitochondria).Cytochrome c released to the cytosol was analyzed by immunoblottingusing Cytochrome c-specific antibodies. Anti-VDAC1 and anti-GAPDHantibodies were used to verify that the cytosolic extracts aremitochondria-free.

Flow Cytometry Using Propidium Iodide (PI) and Annexin V-FITC Staining

Cells, e.g. HeLa cells (2×10³), untreated or treated withapoptosis-inducing reagents, were analyzed for apoptotic cell deathusing PI, annexin V-FITC and flow cytometer analysis. Cells werecollected (1500×g for 10 min), washed, and resuspended in 200 μl bindingbuffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl₂).Annexin V-FITC was added according to the recommended Protocol (EnzoLife Sciences, Switzerland), and the cells were incubated in the darkfor 15 min. Cells were then washed with binding buffer and resuspendedin 200 μl binding buffer, to which PI was added immediately before flowcytometry analysis. At least 10,000 events were collected, recorded on adot plot, and analyzed by the FACSCalibur™ flow cytometer software (BDBiosciences, Franklin Lakes, N.J.).

Preparation of Intermediate 1

Step A

Intermediate 1 was synthesized according to the scheme below.

The starting material reagent 1 (p-trifluoromethoxy-bromobenzene;1-Bromo-4-(trifluoromethoxy)benzene) was used. To a solution of reagent1 (2.41 g, 10 mmol) in toluene (50 mL) were consecutively added thefollowing compounds: reagent 2 (1-Boc-piperazine; tert-butylpiperazine-1-carboxylate) (1.68 g, 9 mmol), Pd₂(dba)₃(tris-(dibenzylidenacetone)dipalladium) (290 mg, 0.5 mmol),2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) (311 mg, 0.5 mmol)and sodium t-butoxide (1.92 g, 20 mmol). The mixture was refluxed underN₂ atmosphere overnight. The solvent was evaporated to provide crudereagent 3 (tert-butyl4-(4-(trifluoromethyl)-phenyl)-piperazine-1-carboxylate) as a residue.

Step B

Reagent 3 was directly used for next step without further purification.

Boc group was removed by acid hydrolysis according to the scheme below

The mixture comprising crude reagent 3 in 50 mL of concentratedhydrochloric acid and 50 mL of dichloromethane was stirred for 1.5 hoursat room temperature. After phase separation, the dichloromethane phasewas discarded, and the aqueous phase was evaporated in vacuo to dryness.The residue was dissolved in 50 mL of aqueous sodium hydroxide solution,2.0 M, and 50 mL dichloromethane were added and stirred for additional1.5 hours. The organic phase was collected and concentrated in vacuo toprovide Intermediate 1 as brown oil (2.0 g, 80% yield for steps A andB).

The Intermediate 1 was analyzed using LC-MS method as described abovewith ramping over 1.6 minutes and elution for 1.4 minutes, withdetection at +246. A representative chromatogram and respective massspectra of two peaks of interest relating to Intermediate 1 arerepresented in FIG. 1.

Example 1 Preparation of compound of Formula 2 (VBIT-3,1-(4-chlorophenyl)-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)pyrrolidine-2,5-dione)

The compound of Formula 2 (VBIT-3) was synthesized according to thereaction scheme below:

To a solution of Intermediate 1 (207 mg, 1 mmol) in methanol (2 mL) wasadded reagent 5 (1-(4-Chlorophenyl)-1H-pyrrole-2,5-dione) (246 mg, 1mmol). The reaction was stirred at room temperature overnight. The finalmixture was concentrated and purified by preparative reverse-phase HPLCto provide compound of Formula 2 (VBIT-3) as white solid (100 mg, 22%yield).

The product (compound of Formula 2 (VBIT-3)) was analyzed using LC-MSmethod as described above with ramping over 3 minutes and elution for 1minute, with detection at +453. The chromatogram is represented in FIG.2 a.

The NMR spectra were obtained on 400 MHz apparatus (by Varian).

¹H NMR (400 MHz, DMSO-d₆: δ7.592 (d, J=2.2 Hz, 2H), 7.339 (d, J=2.2 Hz,2H), 7.201 (d, J=2.2 Hz, 2H), 7.021 (d, J=2.1 Hz, 2H), 4.137 (dd, J=1.3Hz, 1H), 3.159 (m, J=1.1 Hz, 4H), 3.00 (m, J=2.4 Hz, 3H), 2.871 (m,J=1.3 Hz, H), 2.680 (m, J=2.0 Hz, 2H). The spectrum is shown in the FIG.2 b.

Example 2 Preparation of compound of Formula 1 (VBIT-4,N-(4-chlorophenyl)-4-hydroxy-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanamide)

The compound of Formula 1 (VBIT-4) was synthesized according to thereaction scheme below:

Step A

A mixture of Intermediate 1 (2.0 g, 8 mmol) and furan-2(5H)-one(2(5H)-Furanone) (1.3 g, 16 mmol) in methanol (MeOH) (5 mL) was stirredat room temperature overnight. The mixture was evaporated in vacuo andthe residue was purified by reverse phase preparative HPLC to provideIntermediate 2[3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)-dihydrofuran-2(3H)-one)as white solid (1.3 g, 0.4 mmol, 50% yield).

The product (Intermediate 2) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minutes, with detection at +330. The chromatogram is represented in FIG.3.

Step B

To a solution of 4-chloroaniline (254 mg, 2 mmol) in toluene (5 mL)trimethyl aluminum (AlMe₃) was added (2.0 M in toluene, 2 mL). Afterstirring for 10 minutes, Intermediate 2 (330 mg, 1.0 mmol) was added tothe solution and the resulting mixture was heated to 80° C. for 8 hours.After cooling to room temperature, the solvent was evaporated in vacuoand the residue was purified by reverse preparative HPLC to afford thecompound of Formula 1 (VBIT-4) as white solid (200 mg, 44% yield).

The product (Compound of Formula 1) was analyzed using LC-MS method asdescribed above with ramping over 3 minutes and elution for 1 minute,with detection at +457. The chromatogram is represented in FIG. 4 a.

The NMR spectra were obtained on 400 MHz apparatus (by Varian).

¹H NMR (400 MHz, DMSO-d₆): δ10.081 (s, H), 7.601 (d, J=0.5 Hz, 2H),7.341 (d, J=1.2 Hz, 2H), 7.177 (d, J=2.2 Hz, 2H), 6.980 (d, J=2.3 Hz,2H), 4.538 (dd, J=1.2 Hz, 1H), 3.561 (m, J=1.3 Hz, H), 3.440 (m, J=1.4Hz, H), 3.112 (m, J=1.2 Hz, 5H), 2.807 (m, J=1.6 Hz, 2H), 2.709 (m,J=1.5, Hz, 2H), 2.400 (m, J=1.4, Hz, H), 2.150 (m, J=1.4, Hz, H). Thespectrum is shown in the FIG. 4 b.

Example 3 Preparation of compound of Formula 3 (VBIT-12,2-(1-(naphthalen-1-ylmethyl)-4-(phenylamino)piperidine-4-carboxamido)aceticacid)

Step 1

1-(Chloromethyl)naphthalene (8.8 g, 50 mmol) was dissolved indimethylformamide (DMF) (100 mL), and potassium carbonate (13.8 g, 100mmol) was added, followed by 4-piperidone ethylene ketal(1,4-dioxa-8-azaspiro[4.5]decane) (7.2 g, 50 mmol). The mixture wasstirred at room temperature overnight. The solvent was evaporated invacuo and the residue was purified by chromatography in silica gel(eluting with dichloromethane) to provide pure naphthylated ketal(Intermediate 3) as white solid (8.5 g, 60% yield).

Intermediate 3 was analyzed using LC-MS method as described above withramping over 1.6 minutes and elution for 1.4 minute, with detection at+283. The chromatogram is represented in FIG. 5.

Step 2

A solution of Intermediate 3 (product of Step 1) (1.42 g, 5 mmol) in 20ml of 3N hydrochloric acid (HCl) in ethanol (EtOH) was refluxedovernight. The resulting mixture was concentrated in vacuo to provideIntermediate 4 (1-(naphthalen-1-ylmethyl)piperidin-4-one), which wasused with no further purification.

The product (Intermediate 4) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minute, with detection at +239. The chromatogram is represented in FIG.6.

Step 3

Intermediate 4 (N-methylnaphtyl-4-piperidinone) (2.4 g, 10 mmol) andaniline (930 mg, 10 mmol) were dissolved in glacial acetic acid (AcOH)(25 mL). Thereafter, trimethylsilyl cyanide (TMSCN) was added dropwise(1.3 mL, 10 mmol) over a 10-min period, maintaining the temperaturebelow 40° C. using a cold water bath. The solution was stirred overnightand then poured into ammonium hydroxide ice mixture, formed by 50 mL ofconcentrated ammonium hydroxide solution and 100 g of crushed ice.Additional concentrated ammonium hydroxide was slowly added until pHrose to 10. The resultant mixture was extracted three times with 100 mLof chloroform, and the combined organic layers were dried over sodiumsulfate, filtered and concentrated to a yellow nitrile residue(Intermediate 5,1-(naphthalen-1-ylmethyl)-4-(phenylamino)piperidine-4-carbonitrile)which was used in the next step directly without further purification.

The product (Intermediate 5) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minute, with detection at +341. The chromatogram is represented in FIG.7.

Step 4

The nitrile (Intermediate 5) was hydrolyzed according to the schemebelow:

Intermediate 5 (the product of step 3) was mixed with 10 mL ofconcentrated sulfuric acid (H₂SO₄). The mixture was stirred at roomtemperature overnight. A concentrated ammonium hydroxide solution wasslowly added until pH rose to 10. The final mixture was concentrated andpurified by reverse phase preparative HPLC to provide the amide(Intermediate 6,1-(naphthalen-1-ylmethyl)-4-(phenylamino)piperidine-4-carboxamide) aswhite solid (400 mg, 11% yield for the steps 2-4).

The product (Intermediate 6) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minute, with detection at +359. The chromatogram is represented in FIG.8.

Step 5

Intermediate 6 was further hydrolyzed to carboxylic acid according tothe scheme below:

Intermediate 6 (360 mg, 1.0 mmol) was dissolved in ethylene glycol (10mL), and potassium hydroxide (KOH) (280 mg, 5 mmol) was added. Theresulting mixture was heated to 150° C. and stirred overnight. Aftercooling to room temperature, the final mixture was concentrated in vacuoand purified by reverse phase preparative HPLC to provide the freecarboxylic acid (Intermediate 7,1-(naphthalen-1-ylmethyl)-4-(phenylamino)-piperidine-4-carboxylic acid)as white solid (200 mg, 50% yield).

The product (Intermediate 7) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minute, with detection at +360. The chromatogram is represented in FIG.9.

Step 6

Intermediate 7 was glycinated with methyl 2-aminoacetate (methylglycinate) according to the scheme below:

Intermediate 7 (180 mg, 0.5 mmol), HATU(1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]-pyridinium3-oxid hexafluorophosphate) (380 mg, 1.0 mmol),N,N-diisopropylethylamine (DIEA) (260 mg, 2.0 mmol), and methylglycinate (90 mg, 1.0 mmol) were dissolved in dimethyl formamide (DMF)(10 mL), and the solution was stirred at room temperature overnight. Theresulting mixture was concentrated in vacuo and purified by reversephase preparative HPLC to provide the glycinate methyl ester(Intermediate 8, methyl(1-(naphthalen-1-ylmethyl)-4-(phenylamino)piperidine-4-carbonyl)glycinate)as white solid (100 mg, 46% yield).

The product (Intermediate 8) was analyzed using LC-MS method asdescribed above with ramping over 1.6 minutes and elution for 1.4minute, with detection at +431. The chromatogram is represented in FIG.10.

Step 7

Intermediate 8 (the glycinate methyl ester product of Step 6) washydrolyzed with lithium hydroxide in tetrahydrofuran, according to thescheme below:

To a solution of Intermediate 8 (100 mg, 0.23 mmol) in 5 mL of THF, asolution of lithium hydroxide (LiOH) (40 mg, 1.0 mmol) in 5 mL of waterwas added, the resulting mixture was stirred at room temperatureovernight. Thereafter, the pH was adjusted to about 7 with 1.0 N HCl.The mixture was concentrated in vacuo and purified by preparative HPLCto provide the compound of Formula 3 (VBIT-12) (20 mg, 20% yield) aswhite solid.

The product (compound of Formula 3; IUPAC name:2-(1-(naphthalen-1-ylmethyl)-4-(phenylamino)piperidine-4-carbox-amido)aceticacid) was analyzed using LC-MS method as described above with rampingover 3 minutes and elution for 1 minute, with detection at +417. Thechromatogram is represented in FIG. 11 a.

The NMR spectra were obtained on 400 MHz apparatus (by Varian).

¹H NMR (400 MHz, DMSO/D20-d6): δ8.63 (d, H), 8.1 (s, H), 7.9 (d, J=1.2Hz, 2H), 7.89 (d, J=2.2 Hz, 2H), 7.87 (d, J=2.3 Hz, 2H), 7.71 (dd, J=1.2Hz, 2H), 7.67 (d, J=1.3 Hz, 2H), 7.16 (2, J=1.4 Hz, 2H), 6.78 (m, 4H),3.80 (s, 2H), 3.71 (s, 2H), 2.31 (d, J=2.4, Hz, 2H), 2.25 (s, 2H), 2.17(t, J=2.4, 2H), 1.98 (t, J=1.9, 2H), 1.88 (t, J=1.8, 2H)

The spectra in d6-DMSO and in d6-DMSO with D₂O are shown in the FIGS.11b and 11c respectively.

Example 4 Chiral Separation of Compound of Formula 1 (VBIT-4)Enantiomers

Racemic compound of Formula 1 (VBIT-4) was analyzed by analytical chiralHPLC. Briefly, the material was eluted on Chiralpak-IC3 column (4.6×100mm, 3 μm), kept at 35° C., at 2 mL/min, with acetonitrile and 20% of0.1% solution of DEA in methanol. Two peaks, with 0.38 min difference inretention time (2.32 and 2.7 min), were obtained in expected ratio asabout 50.0%. Preparative chiral HPLC was then conducted. Each peak wascollected separately. The enantiomers were analyzed by 400 MHz

NMR but were not discernable in deuterated DMSO.

¹H NMR (400 MHz, DMSO-d₆): δ10.081 (s, H), 7.601 (d, J=0.5 Hz, 2H),7.341 (d, J=1.2 Hz, 2H), 7.177 (d, J=2.2 Hz, 2H), 6.980 (d, J=2.3 Hz,2H), 4.538 (dd, J=1.2 Hz, 1H), 3.561 (m, J=1.3 Hz, H), 3.440 (m, J=1.4Hz, H), 3.112 (m, J=1.2 Hz, 5H), 2.807 (m, J=1.6 Hz, 2H), 2.709 (m,J=1.5, Hz, 2H), 2.400 (m, J=1.4, Hz, H), 2.150 (m, J=1.4, Hz, H).

FIG. 12a demonstrates a representative NMR spectrum in deuterated DMSOrelating to the separated single enantiomer of the compound of Formula1, VBIT-4-1 (also referred to as BGD-4-1). FIG. 12b demonstrates arepresentative NMR spectrum in deuterated DMSO relating to the separatedsingle enantiomer of the compound of Formula 1, VBIT-4-2 (also referredto as BGD-4-2).

Example 5 Bioluminescence Resonance Energy Transfer (BRET-2) Assay forMonitoring Oligomerization of VDAC1 in Living Cells

Plasmids encoding the fusion proteins rat(r)VDAC1-GFP2 and rVDAC1-lucwere constructed using the BRET2 plasmids (Perkin Elmer, Waltham,Mass.). The rVDAC1 gene was cloned into BamHI and HindIII sites of theBRET2 plasmids (N2 variants) and amplified using the forward primerCGAAGCTTATGGCTGTGCCACCCACGTATGCC and the reverse primerGGATCCGCCGCCGCCGGAGCCGCCGCCGCCTGCTTGAAAT-TC. The reverse primer wasdesigned to contain a double linker sequence ((GGGS)₂) connecting VDAC1and the RLuc or GFP₂ genes that introduces flexibility to the region.

A plasmid encoding shRNA against human VDAC1 (hVDAC1) for specificsilencing of endogenous human VDAC1 was introduced into ashRNA-expressing vector. The hVDAC1-shRNA-encoding sequence was createdusing the two complimentary oligonucleotide sequences, each containingthe 19 nucleotide target sequence of hVDAC1 (337-355), followed by ashort spacer and an anti-sense sequence of the target:

oligonucleotide 1,

AGCTTAAAAA CACTAGGCACCGAGATTA TCTCTTGAATAATCTCGGTGC CTAGTGTGand oligonucleotide 2,

GATCC ACACTAGGCACCGAGATTA TTCAAGAGATAATCTCGGTGCCTAG TGTTTTTTA,with the VDAC1-derived sequence being underlined. ThehVDAC1-shRNA-encoding sequence was cloned into the BglII and HindIIIsites of the pSUPERretro plasmid (OligoEngine, Seattle, Wash.),containing a puromycin-resistance gene. Transcription of this sequenceunder the control of the H1 RNA promoter of RNA Polymerase III producesa hairpin (hVDAC1-shRNA).

T-REx-293 cells stably expressing hVDAC1-shRNA, showing low (10 to 20%)endogenous VDAC1 expression (referred to as T-REx-pS10 cells) wereseeded onto 96-well plates at density 9,000 cells per well and incubatedfor at least 24 hours until attached.

The cells were transfected using calcium phosphate method. Transfectionswere carried out with 0.2 μg of a plasmid coding for rVDAC1-Rluc andwith 0.8 μg of a plasmid coding for rVDAC1-GFP2. As a negative control,cells were transfected with plasmids encoding for rVDAC1-Rluc (0.2 μgDNA) and GFP2 (0.8 μg). In another control (control cells), cells werealso transfected with a plasmid encoding for rVDAC1-luc (0.2 μg) andplasmid pcDNA4/TO (0.8 μg).

The BRET2 signal represents the ratio of the GFP2 fluorescence, measuredat its emission wavelength (510 nm), over the light intensity(luminescence) emitted at 395 nm. All measurements were performed usingthe Infinite 200 ELISA reader (Tecan). BRET2 signals were defined asGFP2/Rluc intensity ratio and calculated as follows:

(a) The BRET2 signals obtained in VDAC1-RLuc/pcDNA4/TO cells (controlcells) were subtracted from the signals obtained in cells expressingVDAC1-Rluc and VDAC1-GFP2.

(b) The net ratios of Renilla luciferase and GFP2 activities(GFP2/luciferase ratio after the subtraction of the BRET2 signals fromcontrol cells) were calculated.

(c) The ratios of BRET2 signals between different cells exposed and notexposed to apoptosis inducers were compared.

(d) For validation of BRET2 assay robustness, VDAC1 oligomerization wasinduced by the apoptosis inducer STS (Starosporine). The Z-factor andthe Z′-factor, which is a measure of assay robustness, were calculatedas follows:Z=[BRET2 Ratio(AVG+STS)−(AVG−STS)]/SD(+STS).

A Z factor >3 is considered good, the calculated Z value of about 28 wasobtained.Z′=1−[3×SD(+STS)+3×SD(−STS)]/[AVG(+STS)−AVG(−STS)]

A value of 0.58 for Z′ factor was obtained, which is in the requiredrange of 0.5-1; wherein

SD indicates standard deviation and AVG=average of BRET2 signal ofseveral samples (repeats 10-36).

Alternatively, the Z-factor was calculated using the equation:Z=[BRET2 Ratio(AVG+STS)−(AVG−STS)]/SD(+STS), where a Z factor>3 isconsidered good.

As a measure of the assay robustness, the Z′ factor was obtained usingthe equation: Z′=1−[3×SD(+STS)+3×SD(−STS)]/[AVG(+STS)−AVG(−STS)].

a) A value of 0.58 for Z′ factor, which is in the required range of0.5-1 for cell-based assay.

DNA encoding fusion proteins rVDAC1-Rluc (in which RLuc was connected torVDAC1 at the C terminal position through a linker (GGGS)) andrVDAC1-GFP2 (in which the GFP2 was fused to the rVDAC1 C terminus) wascloned into BRET2 vectors. rVDAC1-GFP2 and rVDAC1-Rluc were expressed inT-REx-293 cells stably expressing shRNA-hVDAC1 and a low level ofendogenous hVDAC1 (referred to as T-REx-pS10 cells) hVDAC1-shRNA, beingspecific to human VDAC1, allowed the expression of rVDAC1 and decreasedthe participation of endogenous hVDAC1 in oligomerization, therebyenhancing the BRET2 signal. rVDAC1-GFP2 and rVDAC1-Rluc expressionlevels were correlated with the amount of plasmids used. Specifically,0.8 μg rVDAC1-GFP2 and 0.1 μg rVDAC1-Rluc were found to give the bestsignal.

The present inventors have recently demonstrated that selenite inducesapoptosis and VDAC1 oligomerization. Therefore selenite was used toenhance BRET-2-detectable VDAC1 oligomerization. Conversely,4,4-diisothiocyanostilbene-trans-2,2-disulfonic acid (DNDS), aninhibitor of VDAC1 channel conductance and apoptosis (Ben-Hail D,Shoshan-Barmatz V.VDAC1-interacting anion transport inhibitors inhibitVDAC1 oligomerization and apoptosis. Biochim Biophys Acta. 2016 July;1863(7 Pt A):1612-2) was used to inhibit any selenite-induced BRET2signal. Chemical cross-linking and Western blot analysis also served todemonstrate any enhancement or inhibition of VDAC1 oligomerization.

T-REx-293 cells were treated as follows: first the cells were incubatedfor 1 hour without or with DNDS at final concentration 200 μM in 100 μL,and then incubated with selenite, at concentration 30 μM, for additional3 hours.

Following incubation, cells were harvested using trypsin, washed twicewith PBS by centrifugation at 1000×g, 5 min, resuspended in 200 μl ofPBS and divided between two wells of a 96-well clear-bottom plate(Grenier). Luciferase activity was assayed using the membrane-permeablesubstrate DBC in PBS supplemented with magnesium chloride (1 g/L) andglucose (1 g/L), with DBC being added to a final concentration of 5 μMjust before luminescence was measured.

The results are shown in the FIG. 13 a.

Example 6 Identification of VDAC1 Oligomerization Inhibitors

The ability of the compounds of the invention to inhibit VDAC1oligomerization was tested by the following methods A and B:

Method A—VDAC1 Oligomerization Induced by Apoptosis-InducingReagents—the BRET2 Assay

The screening was conducted using BRET2 assay as described above.Briefly, T-Rex-293 cells containing low VDAC1 levels, cultured asdescribed above, were transfected to express rVDAC1-GFP2 (0.8 μg) andrVDAC1-Rluc (0.1 μg) and seeded at a density of 9,000 cells/well in a96-well plate. Test compounds were diluted with DMSO to a concentrationof 2 mM of the tested compound and stored frozen. Test compounds (1 μlof 2 mM stock solutions) were added (using a robotic system) to thecells to a final concentration of 10 μM in 100 μl (1% final DMSOconcentration). The cells were pre-incubated for 1 hour with the testedcompounds, and then incubated with one of the following apoptosisinducers: STS, 1 μM (3 h) or selenite, 30 μM (3 h) or As₂O₃, 60 μM(3h)—all in growth medium. After treatment, the medium was removed andassayed for BRET2 signals as described above. Liquid handling was donewith the Tecan (Mannedorf, Switzerland) Freedom 150 Robotic & MCA LiquidHandling System, while luciferase luminescence and fluorescence readingswere obtained a robot-integrated Tecan Infinite M1000 reader.

Method A was used for screening of VDAC1 oligomerization inhibitors. Forexample, drug-like compounds library provided by the National CancerInstitute (NCI) was tested using this method and the results, presentedas the % of inhibition of the BRET2 signal, are shown in the Table 2below.

TABLE 2 Summary of BRET2-based screen results of the anti-VDAC1oligomerization activity of compounds from the NCI library BRET2, % ofinhibition # NSC As₂O₃ Selenite STS 1 16631 0 41.5 30.5 2 48422 0 38.636.8 3 308849 99.5 0 0 4 42537 84.9 0 0 5 324623 78 16.3 0 6 667251 77.50 1.4 7 109292 75.4 0 0 8 31069 74.9 0 0 9 13151 67 0 0 10 163802 65.81.2 0 11 605333 63.7 16 0 12 30205 62.7 0 0 13 205968 58.5 0 0 14 3289255.4 16.4 0 15 10768 52 9.6 0 16 31698 51.3 2.1 0 17 36586 49 0 0 1841066 48.8 0 0 19 39938 48.7 18.8 0 20 151252 100 29.8 0 21 146554 10071.8 0 22 23247 97.3 52.1 0 23 11150 95.2 67 11.6 24 204232 90.3 32.5 025 135618 88.9 64.9 0 26 657149 88.3 25.3 0 27 20045 88 77.4 0 28 26848786.7 69.4 0 29 522131 86.8 47.6 0 30 191029 86.5 36.4 0 31 331208 86.428.3 0 32 28837 85.2 48.3 0.3 33 329249 82.8 22.4 12.1 34 12262 81.567.4 0 35 67436 78.1 65.7 14.8 36 372767 77.3 26.6 0 37 335048 76.5 3517 38 19637 76.2 61.8 0 39 404057 74.6 37.1 0 40 15571 72.3 77.5 1.3 41672441 69.6 41.8 0 42 40275 64.8 35.2 16.8 43 41377 60.4 27.3 0 44 3170356.7 33.3 16.6 45 132868 55.5 41.4 0 46 341956 51.5 24 0 47 8816 49.620.1 0 48 31672 46.4 32.3 17.3 49 317605 46 52 0.3 50 338042 44.7 80.2 051 343966 39.4 20.1 0 52 15362 98 41 39.7 53 601359 97.7 74.4 46 5442199 97 78.7 31 55 10428 96.4 75.7 38.2 56 154389 90.6 90.6 45.9 5719487 88.8 68.6 32.6 58 680515 87.8 66.3 33.4 59 15364 85.4 75.9 40.9 60146771 70.7 68.1 41.2 61 39047 70.2 54 39.1 62 36815 67.7 50 34.5 6319115 64.2 62 36.4 64 319990 96.2 43.2 22 65 43678 95.1 78.3 34.6 66252172 83.1 50 44.1 67 103520 82.5 74 22.7 68 43344 80.7 50.1 23.8 69372275 72.5 46.5 28.5 70 41376 71.6 41.8 29.7 71 321502 67.3 46.5 22.4

Table 2 provides a summary of BRET2-based screen results of theanti-VDAC1 oligomerization activity of compounds from the NCI library.Results are presented as percent inhibition of the BRET2 signal inducedby the indicated pro-apoptotic agent. The twelve most active compoundsfrom the NCI library, with the three inducers, as identified by thistest, were compounds number: 15362, 601359, 42199, 10428, 154389, 19487,680515, 15364, 146771, 39047 and 19115.

Method B—VDAC1 Oligomerization Assayed by Chemical Cross-Linking

T-Rex-293 cells were treated as described in Example 5 hereinabove, andcross-linked with EGS as described above in Methods (VDAC1cross-linking), subjected to SDS-electrophoresis and immunoblotted forVDAC1. The results are shown in the FIG. 13 b.

The present inventors have previously reported the equivalence of themethod A and B for apoptosis inhibitor4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS) (Ben-Hail D,Shoshan-Barmatz V. VDAC1-interacting anion transport inhibitors inhibitVDAC1 oligomerization and apoptosis. Biochim Biophys Acta. 2016 1863(2016) 1612-1623).

The ability of the compounds of the invention to inhibit VDAC1oligomerization was tested by Method B and the results are provided inExamples 8-12 hereinafter.

Example 8 VDAC1 Oligomerization Inhibition by Racemic VBIT-4 andEnantiomers

HEK-293 cells were incubated with racemic compound of Formula 1(VBIT-4), enantiomer 1 of compound of Formula 1 (VBIT-4-1 (alsoidentified as BGD-4-1)) or enantiomer 2 of compound of Formula 1(VBIT-4-2 (also identified as BGD-4-2)) (10 μM) for 2 h and then with orwithout selenite (15 μM, 4 h). The cells were harvested, cross-linkedwith EGS (300 μM, 15 min) as described above, and analyzed by immunoblotusing anti-VDAC1 antibodies.

The results are presented in the FIG. 14a . The positions of VDAC1monomers and multimers are indicated. The star indicates monomeric VDAC1with modified electrophoretic mobility, representing intra-molecularcrossed-linked monomeric VDAC1.

Example 9 Racemic Compound of Formula 1 (VBIT-4) and Enantiomers ofCompound of Formula 1 (VBIT-4-1 and VBIT-4-2) Inhibit Apoptotic CellDeath

HeLa cells were incubated with varying concentrations (2-20 μM) ofracemic compound of Formula 1 (VBIT-4) and of the enantiomers ofcompound of Formula 1 (VBIT-4-1 and VBIT-4-2) for 1 hour, and then withor without selenite (25 μM, 3 hours). Cells were harvested and assayedfor apoptotic cell death, using PI staining and FACS analysis. Theresults shown in the FIG. 14b correspond to means±SD (n=3).

Example 10 Inhibition of VDAC1 Oligomerization, Apoptosis and CytochromeC Release by Compound of Formula 10 (AKOS-022), Compound of Formula 2(VBIT-3) and Racemic Compound of Formula 1 (VBIT-4), in HEK-293 Cells

a. HEK-293 cells were incubated without and with the following testedcompounds: compound of Formula 10 (AKOS-022), compound of Formula 2(VBIT-3) or racemic compound of Formula 1 (VBIT-4) (2.5-15 μM) for 2hours and then with or without selenite (15 μM, 4 h), trypsinized,washed with PBS, protein concentration was determined and harvested,cross-linked with EGS (3 mg protein/ml, 300 μM, 15 min), and analyzed byimmunoblot using anti-VDAC1 antibodies. The positions of VDAC1 monomersand multimers are indicated in the FIG. 15a . The star indicatesmonomeric VDAC1 with modified electrophoretic mobility, representingintra-molecular crossed-linked monomeric VDAC1.

Quantitative data of the selenite-induced VDAC1 dimer formation by thetested compounds is presented as inhibition percentile, in the FIG. 15b. The results show means±SD (n=3). The closed circle (●) indicatesVBIT-4 (compound of Formula 1), the open circle (∘) indicates compoundof Formula 10, and an open square (□) indicates VBIT-3 (compound ofFormula 2).

b. Additionally, the inhibition of selenite-induced apoptosis by thecompounds as analyzed using annexin V-FITC/PI staining and FACS,presented in the FIG. 15 c.

c. Cytochrome c (Cyto c) release was determined as described above inMethods (Cytochrome c release from mitochondria). Briefly, to assessCyto c release, cells were incubated on ice for 10 min with 0.025%digitonin, centrifuged, and the pellet (mitochondria—Mito) andsupernatants (cytosol—Cytos) were subjected to SDS-PAGE andimmunoblotting, using anti-Cyto c, antibodies. Anti-VDAC1 and anti-GAPDHantibodies were used to verify that the cytosolic extracts aremitochondria-free. The results of Cyto c release from the mitochondriaas induced by selenite are presented as immunoblots, with the cytosolicand mitochondrial fractions confirmed by immunoblotting of GAPDH(glyceraldehyde-3-phosphate dehydrogenase) and VDAC1, respectively, inthe FIG. 15d . The quantitative data of selenite-induced Cyto c releaseto the cytosol by the tested compounds is presented in the FIG. 15e .The data are presented as percent inhibition. The results showncorrespond to means±SD (n=3). The closed circle (●) indicates VBIT-4(compound of Formula 1), the open circle (∘) indicates compound ofFormula 10 (AKOS-022), and an open square (□) indicates VBIT-3 (compoundof Formula 2).

d. IC50 values of the tested compounds were derived from the obtaineddata. The results shown correspond to means±SD (n=3).

TABLE 3 VDAC1 Cyto c Compound oligomerization release Apoptosis Formula# (name) IC₅₀, μM IC₅₀, μM IC₅₀, μM Formula 10 (AKOS 022) 3.3 ± 0.18 3.6± 0.4  3.4 ± 0.2  Formula 2 (VBIT-3) 8.8 ± 0.56 6.6 ± 1.03 7.5 ± 0.27Formula 1 (VBIT-4) 1.9 ± 008  1.8 ± 0.24 2.9 ± 0.12

Example 11 Inhibition of VDAC1 Oligomerization and of Apoptosis, inNeuronal Cells and in Bax/Bak-Lacking Cells, by Compound of Formula 10(AKOS-022) and Racemic Compound of Formula 1 (VBIT-4)

SH-SY5Y cells and Bax^(−/−)/Bak^(−/−) MEFs cells were incubated with thefollowing tested compounds: compound of Formula 10 (AKOS-022) or racemiccompound of Formula 1 (VBIT-4) (30 μM, 2 h), and then with or withoutcisplatin (20 μM, 20 h). The cells were harvested, cross-linked with EGS(200 μM, 15 min), and analyzed by immunoblot using anti-VDAC1antibodies.

The results from SH-SY5Y cells are presented in the FIG. 16a . Theresults from Bax^(−/−)/Bak^(−/−) MEFs cells are presented in the FIG.16b . The positions of VDAC1 monomers and multimers are indicated. Thestar indicates monomeric VDAC1 with modified electrophoretic mobility,representing intra-molecular crossed-linked monomeric VDAC1.

Quantitative analysis of cisplatin-induced VDAC1 dimers formation (graycolumns) and apoptosis as analyzed using annexin V-FITC/PI staining andFACS (black columns) in SH-SY5Y cells, in the absence and presence ofthe tested compounds, is presented in the FIG. 16c . The results showncorrespond to mean±SD (n=3), p<0.001(***).

Quantitative analysis of cisplatin-induced VDAC1 dimers formation andcytochrome c release by the tested compounds in Bax^(−/−)/Bak^(−/−) MEFscells is presented in the FIG. 16 d.

Example 12 Correlation Between the Extent of Inhibition of Apoptosis andof VDAC1 Oligomerization by Compound of Formula 10 (AKOS-022)

HeLa cells were incubated with compound of Formula 10 (AKOS-022) at 0-20μM for 2 hours and further with or without selenite (30 μM, 3 h) orcisplatin (15 μM, 20 h). Cells were harvested and either cross-linkedwith EGS (300 μM, 15 min) as described above, and analyzed for VDAC1oligomerization by immunoblot using anti-VDAC1 antibodies, or assayedfor apoptotic cell death using annexin V-FITC/PI staining and FACSanalysis. The gel of selenite experiment is represented in the FIG. 17a.

Quantitative analysis of the inhibition of VDAC1 dimer levels and cellsundergoing apoptosis as induced by selenite, as a function of compoundof Formula 10 (AKOS-022) concentration, are presented in the FIG. 17b .The results reflect means±SD (n=3).

The gel of cisplatin experiment is represented in the FIG. 17c .Quantitative analysis of the inhibition of VDAC1 dimer levels and cellsundergoing apoptosis as induced by cisplatin, as a function of compoundof Formula 10 (AKOS-022) concentration, are presented in the FIG. 17d .The results reflect means±SD (n=3).

Quantitative analysis of the extent of apoptosis inhibition as afunction of the inhibition of VDAC1 dimer formation is presented in FIG.17e . Apoptosis was induced either by selenite (solid square—▪) orcisplatin (empty square—□), and was analyzed at the identical AKOS-022concentration as described above.

Example 13 Interaction of Tested Compounds with Purified VDAC1 and withLipid Bilayer-Reconstituted Purified VDAC1 and Reduced ChannelConductance

VDAC1 was purified as described above in Methods (VDAC1 purification).Purified VDAC1 was reconstituted into a planar lipid bilayer (PLB)membrane and currents through VDAC1, in response to a voltage step from0 to 10 mV, were recorded before and 30 min after the addition of 40 μMof the following test compounds: compound of Formula 10 (AKOS-022),compound of Formula 2 (VBIT-3) or racemic compound of Formula 1(VBIT-4), as shown in the FIG. 18 a.

Additionally, the channel conduction via multi-channel recordings asfunction of voltage, and the average steady-state conductance of VDAC1were measured.

FIG. 18b demonstrates channel conductance before (solid squares) and 30min after the addition of compound of Formula 10 (AKOS-022) (emptycircle), of compound of Formula 2 (VBIT-3) (empty square), or of racemiccompound of Formula 1 (VBIT-4) (solid circle). Relative conductance(conductance/maximal conductance) was determined at a given voltage. Thedata were normalized according to the conductance at −10 mV (maximalconductance).

Example 14 Binding Affinities of Tested Compounds to Purified VDAC1

Purified VDAC1 (133 nM), labeled using the NanoTemper fluorescentprotein-labeling Kit BLUE (Nano Temper technologies, Munich, Germany),according to the manufactory instructions, was incubated with increasingconcentrations of the following tested compounds: compound of Formula 10(AKOS-022), compound of Formula 2 (VBIT-3), or racemic compound ofFormula 1 (VBIT-4). After 20 min of incubation, the samples (3-5 μL)were loaded into MST-grade glass capillaries (Monolith NT Capillaries),and the thermophoresis process was measured using the Monolith-NT115apparatus. The results are presented in the FIG. 18c as % of the boundfraction, of compound of Formula 10 (AKOS-022) (open circle), ofcompound of Formula 2 (VBIT-3) (open square), or of racemic compound ofFormula 1 (VBIT-4) (closed circle) (0.3 μM to 100 μM), each withpurified VDAC-1.

The fraction bound was calculated as:

${{Fraction}\mspace{14mu}{bound}} = {100 \times \frac{F - {Fmin}}{{Fmax} - {Fmin}}}$

Fmax and Fmin represent the maximal and minimal fluorescence,respectively and F the fluorescence measured in the presence of thetested compound.

VDAC1 binding affinities of tested compounds were calculated from theMST measurements. The results of means±SD (n=3) are 15.4±2.9 μM for thecompound of Formula 10 (AKOS022), 31.3±1.7 μM for the compound ofFormula 2 (VBIT-3), and 17±5.3 μM for the racemic compound of Formula 1(VBIT-4).

Example 15 The Effect of the Tested Compounds on Selenite-InducedIncreases in Intracellular Calcium Levels, Mitochondrial MembranePotential and Reactive Oxygen Species (ROS) Levels

a. HEK-293 cells were incubated with the following test compounds:compound of Formula 10 (AKOS-022), compound of Formula 2 (VBIT-3), orcompound of Formula 1 (VBIT-4) (15 μM, 2 hours) and then with or withoutselenite (15 μM, 4 hours). The cells were harvested and intracellularcalcium ([Ca²⁺]i) levels were measured using Fluo-4 and FACS analysis,as described above. Quantitative analysis of the results as percentileof maximal [Ca²⁺]i is presented in the FIG. 19 a.

b. Mitochondrial membrane potential (ΔΨ) was analyzed with TMRM and FACSanalysis. CCCP (25 μM, 30 min) served as a positive control formitochondrial AT) dissipation and the CCCP-sensitive TMRM fluorescence.The respective results are presented in FIG. 19 b.

c. Cellular ROS levels were analyzed with carboxy-H2DCFDA and FACSanalysis. Mitochondrial superoxide was detected with MitoSOX Red andflow cytometry. The respective results are presented in FIGS. 19c -d.

All the results shown in FIGS. 19a-d correspond to mean±SD (n=3),p<0.05(*), <0.01 (**) or <0.001(***).

Example 16 Preparation of PLGA Nanoparticles of Compound of Formula 10

About 10 mg of AKOS-022 was dissolved in 1 mL of acetone, followed by 50mg of PLGA. This organic phase mixture was added in a drop-wise manner(ca. 0.5 ml/min) to 20 ml of aqueous solution containing 1% polyvinylalcohol (PVA) (w/v) as the stabilizer. The mixture was then stirred at400 rpm by a laboratory magnetic stirrer at room temperature untilcomplete evaporation of the organic solvent. The redundant stabilizerwas removed from the nanoparticles dispersion by centrifugation at15,000×g at 4° C. for 20 min. The pellet was re-suspended in steriledouble distilled water and washed three times.

Blank nanoparticles were prepared in the same manner except AKOS-022addition.

Example 17 Preparation of PLGA Nanoparticles of Compound of Formula 1

PLGA nanoparticles containing the compound of Formula 1 (VBIT-4) wereprepared according to the Example 16, with 10 mg of VBIT-4 substituting10 mg of AKOS022.

Example 18 Brain Penetration of and Exposure to the Compounds ofFormulae 1 and 10

C57BL/6 mice (20 gm) were used. The animals received treatments witheither free compounds of Formulae 1 and 10, or encapsulated compounds,prepared according to the Examples 16 and 17. Doses as indicated in theTable 4 below were administered in phosphate-buffered saline (PBS)through an oral gavage. After 12 hours and 24 hours, randomly selectedmice from each group were sacrificed, their brains collected and storedin −80° C. The concentrations of the compounds in the tissues weredetermined by HPLC/MS analysis. Tissue samples were homogenized inphosphate-buffered saline solution, then diluted with acetonitrile to50% v/v, centrifuged at 10,000 g for 10 minutes, and supernatants wereanalyzed. The compounds concentrations in the brain extracts weredetermined from calibration curves. The results were the averaged from 2mice for each treatment group/time point.

TABLE 4 Compounds in brain Compound Treatment extract, μM Formula 10 50mg/kg, 12 h  4.2 ± 0.714 Formula 10 50 mg/kg, 24 h 1.24 ± 0.23  Formula10 50 mg/kg in PLGA, 12 h 4.36 ± 0.148 Formula 10 50 mg/kg in PLGA, 24 h2.8 ± 1.19 Formula 1 50 mg/kg in PLGA, 24 h 0.190 ± 0.07  Formula 1 50mg/kg, 24 h 0.120 ± 0.02 

The compounds of Formulae 1 and 10 (AKOS-022 and VBIT-4) given orallyreached the brain both when giving in solution or encapsulated in PLGAnano-particles. However, while 12 hours after administration the levelof AKOS-022 in the brain was similar in both cases, after 24 hours thelevel of AKOS022 or VBIT-4 was over 2-fold when encapsulated in PLGA.The AKOS-022 and VBIT-4 levels in the brain is in its effective range(IC50=1 μM). Thus the molecules when administrated either non- orPLGA-encapsulated can reach the brain.

Example 19 Effect of Compound of Formula 1 on Learning and Memory Taskof 5XFAD Transgenic Mice with Alzheimer Disease-Like Disease Using theRadial-Arm Water Maze for Testing Learning and Memory Task

The effect of the compound of Formula 1 (VBIT-4) on learning and memorytask of 5XFAD transgenic mice with AD-like disease was tested asdisclosed in Webster, S. J., et al, (2014) Frontiers in genetics 5, 88,([5XFAD B6.Cg-Tg APPSwFlLon, PSEN1*M146Ln*L286V6799Vas/J]). These micepresent detectable phenotypes of intracellular and extracellular amyloidplaques at 2 months of age, develop cognitive impairments at 4-5 monthsand exhibits neuronal death at 9 months.

The compound of Formula 1 (VBIT-4) was dissolved in drinking water asfollows. About 24 mg of VBIT-4 were transfer to Eppendorf tube anddissolved in 120 μl of 100% DMSO by Vortex mixer. Clear solution wasobtained. Solution of 1 M of HCl, about 10 mL, was prepared from 6-M HClsolution, provided by Pierce, Rockford, Ill., USA. About 370 μL of the1M HCl solution was used to acidify 120 mL of drinking water. The VBIT-4DMSO solution (120 μL) was slowly added (by dropping) into the acidicwater and mixed by magnetic stirring. The final pH was between 4.8 and5.0. If the solution became milky, further 10 to 30 μL of HCl solutionwere added to obtain clear solution. The amount was sufficient for 24mice at dose of 20 mg/kg and drinking volume of 5 mL per mouse per day.

Animals at age two months were assigned to three groups: transgenictreated (TG-T, 8 males and 3 females), transgenic vehicle (TG-V, 8 malesand 3 females), and wild type (WT, 10 males and 8 females). Of these, 2males in TG-T group dies during the study.

Two-months old 5XFAD mice were provided with 0.9% of DMSO solution orVBIT-4 solution (20 mg/kg in 0.9% DMSO) in drinking water replaced withfresh solution three times a week in the first month and thereaftertwice a week for additional 3 months.

When the mice reached the age of six months, a two-day radial-arm watermaze (RAWM) trial was performed as described previously (JenniferAlamed, et al, Nature Protocols 1, (2006) 1671-1679) to test the effectof VBIT-4 on learning and memory task. The RAWM containing six swimpaths (arms) was used. The arms were extending out from an open centralarea with an escape platform located at the end of one arm (the goalarm). The goal arm location remained constant for a given mouse. On day1, mice were trained for 15 trials (spaced over 3 h), with trialsalternating between visible and hidden platforms. On day 2, mice weretrained for 15 trials with the hidden platform.

Entry into an incorrect arm was scored as an error, and the times spentby the animal to find the platform were recorded. The results aredemonstrated in the FIGS. 20A and 20B. FIG. 20A shows the number oferrors, while FIG. 20B shows the total time spend in the water maze, asfunction of number of learning blocks. The number of errors data (FIG.20A, data presented as mean±standard error of the mean) from RAWM wasassessed using ANOVA test; a significant difference in animal's memorytraining between the three groups: WT-mice (n=18), 5XFAD/APOE (n=13) and5XFAD/APOE treated with VBIT-4 (n=9) in different measurement times(trials) was obtained with F (9,159)=2.03 (p=0.03). To examine thesource of differences the post-hoc test of Bonferroni-type was used. Thenon-transgenic mice trained better in comparison to non-treated TG mice(p=0.007). The TG mice treated with VBIT-4 performance was better thanuntreated TG and there was no difference between TG VBIT-4 treated groupand the WT group. A trend for the improved performance (training) of theVBIT-4-TG treated group compared to non-treated TG group was seen(p=0.06).

The data of total time spent (FIG. 20B, data presented as mean±standarderror of the mean) from RAWM was analyzed using repeated measures ANOVAtest; a significant difference was obtained in animal's memory trainingbetween the three mouse groups: WT (n=18), 5XFAD/APOE (n=13) and5XFAD/APOE treated with VBIT-4 (n=9) in different measurement times(trials), with F (2,35)=6.91, p=0.003. To examine the source ofdifferences the post-hoc test of Bonferroni-type was used. Thenon-transgenic mice trained learnt better in comparison to non-treatedTG mice (p=0.003). The performance of TG mice treated with VBIT-4 wasbetter than untreated TG and there was no difference between TG VBIT-4treated group and the WT group.

The invention claimed is:
 1. A compound of general formula (Id):

wherein L² is a linking group selected from the group consisting of4-hydroxybutanamidylene having the formula HO—CH₂—C*H—CH₂—C(O)NH—,wherein the asterisk denotes an attachment point andpyrrolidine-2,5-dionylene; Z is C₁₋₂-perfluoroalkoxy, and Y is halogen;or an enantiomer, diastereomer, mixture or salt thereof.
 2. The compoundof claim 1 having Formula 1:

or an enantiomer, diastereomer, mixture or salt thereof.
 3. The compoundof claim 1 having Formula 2:

or an enantiomer, diastereomer, mixture or salt thereof.
 4. Apharmaceutical composition containing the compound according to claim 1together with one or more pharmaceutically acceptable excipients.
 5. Amethod of making a medicament comprising formulating a compoundaccording to claim 1 with one or more pharmaceutically acceptableexcipients.
 6. A method for treating a disease or disorder associatedwith enhanced apoptosis comprising administering to a subject in needthereof a therapeutically effective amount of the compound according toclaim
 1. 7. A method for treating a disease or disorder selected fromthe list consisting of neurodegenerative diseases and disorders,cardiovascular diseases and disorders, Alzheimer's disease, Parkinson'sdisease, cardiac hypertrophy, heart failure, myocardial infarction,ischemia/reperfusion injury, apoptosis, autophagy of cardiac myocytes,atrial fibrillation (AF), and cardiac arrhythmia comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the compound according to claim 1.